Methods for detecting AAV

ABSTRACT

Provided herein are methods for determining the serotype of a virus particle and/or or determining the heterogeneity of a virus particle (e.g., an AAV particle). In other embodiments, the invention provides methods to determine the heterogeneity of AAV particles. In some aspects, the invention provides viral particles (e.g., rAAV particles) with improved stability and/or improved transduction efficiency by increasing the acetylation and/or deamidation of capsid proteins.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Phase application under 35 U.S.C. § 371of International Application No. PCT/US2017/046814, filed Aug. 14, 2017,which claims the priority benefit of U.S. Provisional Application No.62/375,314, filed Aug. 15, 2016, the disclosure of each of which ishereby incorporated by reference in its entirety.

SEQUENCE LISTING

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 159792014100SEQLIST.TXT,date recorded: Feb. 13, 2019, size: 52 KB).

FIELD OF THE INVENTION

The present invention relates to methods for serotyping and/ordetermining the heterogeneity of a viral particle (e.g., anadeno-associated virus (AAV) particle) using mass determination, e.g.,by employing liquid chromatography/mass spectrometry (LC/MS) or liquidchromatography/mass spectrometry-mass spectrometry (LC/MS/MS). In someaspects, the present invention relates to methods to improve thestability of AAV particles.

BACKGROUND OF THE INVENTION

Complete characterization of the viral capsid proteins of viral vectors(e.g., AAV vectors), including their sequence and post-translationmodifications, is desired in gene therapy research and development sinceviral capsid proteins (VPs) are critical for viral infectivity.

Viral vector products such as recombinant Adeno-Associated Virus (rAAV)products are typically identified using molecular tools targeting thenucleic acid transgene. These methods may include polymerase chainreaction (PCR) targeting transgene-specific sequences and RestrictionFragment Length Polymorphism (RFLP) techniques. As rAAV technologiesevolve, many facilities are beginning to investigate multiple AAV capsidserotypes encoding their therapeutic transgene in an effort to improvetargeted tissue tropism.

Traditional molecular identification methods identify productscontaining unique transgenes but are unable to discern those that havediffering AAV capsid serotypes. Currently, most AAV serotype identitytests are based on SDS-PAGE banding patterns, an antibody-based ELISA,or a Western blot assay. However, the banding patterns and antibodiesare not specific enough to differentiate different AAV serotypes.Gel-LC/MS/MS has been reported as a capsid serotype identificationmethod. However, this method involves multiple steps including SDS-PAGE,in-gel digestion, and LC/MS/MS and thus requires multiple days for theanalysis while providing limited sequence coverage. Methods foridentifying vectors such as rAAV vectors are of interest to gene therapyvectors (see, e.g., U.S. PG Pub. No. US20110275529). Thus, it would beuseful to have improved methods of characterizing viral particles.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

Using rAAV as an example, described herein is the use of LC/MS as ananalytical tool to specifically identify different viral capsidserotypes (e.g., rAAV capsid serotypes). As part of viralcharacterization, LC/MS can be used to augment the molecularidentification methods. This analytical combination can satisfyregulatory requirements by discerning both the identity of the product'stherapeutic transgene and the identity of the capsid serotype. Thismethod can be used e.g., as an AAV serotype identity test or to monitorviral capsid protein heterogeneity in recombinant AAV gene therapydevelopment. It can also be used to confirm VP sequences in capsidengineering research. In addition, this technique can be used to studythe impact of post translation modifications, such as N terminalacetylation of viral capsid proteins, on transfection potency andintracellular protein trafficking.

The methods described herein can also be used to design AAV particlesfor greater stability and/or improved transduction efficiency; forexample by altering the amino acid residue at position 2 of VP1 and/orVP3 of the AAV capsid such that the amino acid at position 2 isacetylated to a higher extent compared to a wild type AAV capsid. Insome embodiments, the methods can be used to design AAV particles withreduced transduction efficiency; for example by altering the amino acidresidue at position 2 of VP1 and/or VP3 of the AAV capsid such that theamino acid at position 2 is deacetylated to a higher or lower extentcompared to a wild type AAV capsid.

In some aspects, the invention provides a method to determine theserotype of a viral particle comprising a) denaturing the viralparticle, b) subjecting the denatured viral particle to liquidchromatography/mass spectrometry (LC/MS), and c) determining the massesof one or more capsid proteins of the viral particle; wherein thespecific combination of masses of the one or more capsid proteins areindicative of the virus serotype. In some embodiments, the calculatedmasses of the one or more capsid proteins are compared to thetheoretical masses of the one or more capsid proteins of one or morevirus serotypes.

In some aspects, the invention provides a method of determining theheterogeneity of a viral particle comprising a) denaturing the viralparticle, b) subjecting the denatured viral particle to liquidchromatography/mass spectrometry/mass spectrometry (LC/MS/MS), c)determining the masses of one or more capsid proteins of the viralparticle, and d) comparing the masses of step c) with the theoreticalmasses of the one or more capsid proteins of the virus serotype; whereina deviation of one or more of the masses of the one or more capsidproteins are indicative of the viral capsid heterogeneity. In someembodiments, the heterogeneity comprises one or more of mixed serotypes,variant capsids, capsid amino acid substitutions, truncated capsids, ormodified capsids.

In some embodiments of the above aspects, the liquid chromatography isreverse phase liquid chromatography, size exclusion chromatography,hydrophilic interaction liquid chromatography, or cation exchangechromatography. In some embodiments, the viral particle comprises aviral vector encoding a heterologous transgene.

In some aspects, the invention provide a method to determine theserotype of a viral particle comprising a) denaturing the viralparticle, b) subjecting the denatured viral particle to reduction and/oralkylation, c) subjecting the denatured viral particle to digestion togenerate fragments of one or more capsid proteins of the viral particle,d) subjecting the fragments of the one or more capsid proteins to liquidchromatography/mass spectrometry-mass spectrometry (LC/MS/MS), and e)determining the masses of fragments of the one or more capsid proteinsof the viral particle; wherein the specific combination of masses offragments of the one or more capsid proteins are indicative of the viralserotype. In some embodiments, the calculated masses of the fragments ofthe one or more capsid proteins are compared to the theoretical massesof fragments of the one or more capsid proteins of one or more viralserotypes.

In some aspects, the invention provides a method of determining theheterogeneity of a serotype of a viral particle comprising a) denaturingthe viral particle, b) subjecting the denatured viral particle toreduction and/or alkylation, c) subjecting the denatured viral particleto digestion to generate fragments of one or more capsid proteins of theviral particle, d) subjecting the fragments of the one or more capsidproteins to liquid chromatography/mass spectrometry-mass spectrometry(LC/MS/MS), e) determining the masses of fragments of the one or morecapsid proteins of the viral particle, and f) comparing the masses ofstep e) with the theoretical masses of fragments of the one or morecapsid proteins of the viral serotype; wherein a deviation of one ormore of the masses of the one or more capsid proteins are indicative ofthe viral capsid heterogeneity. In some embodiments, the heterogeneitycomprises one or more of mixed serotypes, variant capsids, capsid aminoacid substitutions, truncated capsids, or modified capsids. In someembodiments, the liquid chromatography is reverse phase liquidchromatography, size exclusion chromatography, hydrophilic interactionliquid chromatography, or cation exchange chromatography.

As shown herein the methods can be performed in the absence of a gelseparation step (e.g., sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE)).

In some embodiments of the above aspects and embodiments, the viralparticle comprises a viral vector encoding a heterologous transgene. Insome embodiments, the viral particle belongs to a viral family selectedfrom the group consisting of Adenoviridae, Parvoviridae, Retroviridae,Baculoviridae, and Herpesviridae. In some embodiments, the viralparticle belongs to a viral genus selected from the group consisting ofAtadenovirus, Aviadenovirus, Ichtadenovirus, Mastadenovirus,Siadenovirus, Ambidensovirus, Brevidensovirus, Hepandensovirus,Iteradensovirus, Penstyldensovirus, Amdoparvovirus, Aveparvovirus,Bocaparvovirus, Copiparvovirus, Dependoparvovirus, Erythroparvovirus,Protoparvovirus, Tetraparvovirus, Alpharetrovirus, Betaretrovirus,Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, Lentivirus,Spumavirus, Alphabaculovirus, Betabaculovirus, Deltabaculovirus,Gammabaculovirus, Iltovirus, Mardivirus, Simplexvirus, Varicellovirus,Cytomegalovirus, Muromegalovirus, Proboscivirus, Roseolovirus,Lymphocryptovirus, Macavirus, Percavirus, and Rhadinovirus.

In some aspects, the invention provides a method to determine theserotype of an adeno-associated virus (AAV) particle comprising a)denaturing the AAV particle, b) subjecting the denatured AAV particle toliquid chromatography/mass spectrometry (LC/MS), and c) determining themasses of VP1, VP2 and VP3 of the AAV particle; wherein the specificcombination of masses of VP1, VP2 and VP3 are indicative of the AAVserotype. In some embodiments, the calculated masses of VP1, VP2 and VP3are compared to the theoretical masses of VP1, VP2 and VP3 of one ormore AAV serotypes.

In some aspects, the invention provides a method of determining theheterogeneity of an AAV particle comprising a) denaturing the AAVparticle, b) subjecting the denatured AAV particle to liquidchromatography/mass spectrometry/mass spectrometry (LC/MS/MS), c)determining the masses of VP1, VP2 and VP3 of the AAV particle, and d)comparing the masses of step c) with the theoretical masses of VP1, VP2and VP3 of the AAV serotype; wherein a deviation of one or more of themasses of VP1, VP2 or VP3 are indicative of the AAV capsidheterogeneity. In some embodiments, the heterogeneity comprises one ormore of mixed serotypes, variant capsids, capsid amino acidsubstitutions, truncated capsids, or modified capsids.

In some embodiments of the above aspects and embodiments, the AAVparticle is denatured with acetic acid, guanidine hydrochloride and/oran organic solvent. In some embodiments, the liquid chromatography isreverse phase liquid chromatography, size exclusion chromatography,hydrophilic interaction liquid chromatography, or cation exchangechromatography. In some embodiments, the liquid chromatography isreverse phase liquid chromatography. In some embodiments, the reversephase chromatography is a C4 or C8 reverse chromatography. In someembodiments, the chromatography uses a mobile phase A comprising formicacid in water. In some embodiments, the mobile phase A comprises about0.1% formic acid. In some embodiments, the chromatography comprises amobile phase B comprising formic acid in acetonitrile. In someembodiments, the mobile phase B comprises about 0.1% formic acid. Insome embodiments, the proportion of mobile phase B in the chromatographyincreases over time. In some embodiments, the proportion of mobile phaseB in the chromatography increases in a stepwise manner. In someembodiments, mobile phase B increases from about 10% to about 20%, fromabout 20% to about 30%, and from about 30% to about 38%. In someembodiments, mobile phase B increases from about 10% to about 20% inabout 6 minutes, from about 20% to about 30% in about 10 minutes, andfrom about 30% to about 38% in about 40 minutes. In some embodiments,the liquid chromatography is ultra-performance liquid chromatography(UPLC).

In some embodiments of the above aspects and embodiments, the massspectrometry comprises a capillary voltage of about 3.5 kV. In someembodiments, the mass spectrometry comprises a sampling cone voltage ofabout 45 V. In some embodiments, the mass spectrometry comprisesassisted calibration. In some embodiments, sodium iodide is used as acalibrant.

In some embodiments of the above aspects and embodiments, the N-terminusof VP1 and/or VP3 is acetylated. In some embodiments, the AAV particleis a recombinant AAV (rAAV) particle. In some embodiments, the AAVparticle comprises an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, anAAV4 capsid, an AAV5 capsid, an AAV6 capsid, an AAV7 capsid, an AAV8capsid, an AAVrh8 capsid, an AAV5 capsid, an AAV10 capsid, an AAVrh10capsid, an AAV11 capsid, an AAV12 capsid, an AAV LK03 capsid, anAAV2R471A capsid, an AAV2/2-7m8 capsid, an AAV DJ capsid, an AAV DJ8capsid, an AAV2 N587A capsid, an AAV2 E548A capsid, an AAV2 N708Acapsid, an AAV V708K capsid, a goat AAV capsid, an AAV1/AAV2 chimericcapsid, a bovine AAV capsid, or a mouse AAV capsid rAAV2/HBoV1 (chimericAAV/human bocavirus virus 1). In some embodiments, the AAV capsidcomprises a tyrosine mutation or a heparin binding mutation. In someembodiments, the masses of VP1, VP2, and VP3 are compared to thetheoretical masses of one or more of AAV1 capsid, an AAV2 capsid, anAAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid, an AAV7capsid, an AAV8 capsid, an AAVrh8 capsid, an AAV9 capsid, an AAV10capsid, an AAVrh10 capsid, an AAV11 capsid, an AAV12 capsid, an AAV LK03capsid, an AAV2R471A capsid, an AAV2/2-7m8 capsid, an AAV DJ capsid, anAAV DJ8 capsid, an AAV2 N587A capsid, an AAV2 E548A capsid, an AAV2N708A capsid, an AAV V708K capsid, a goat AAV capsid, an AAV1/AAV2chimeric capsid, a bovine AAV capsid, or a mouse AAV capsid rAAV2/HBoV1(chimeric AAV/human bocavirus virus 1), an AAV2HBKO capsid, an AAVPHP.Bcapsid or an AAVPHP.eB capsid.

In some embodiments of the above aspects and embodiments, the viralparticle comprises an AAV1 ITR, an AAV2 ITR, an AAV3 ITR, an AAV4 ITR,an AAV5 ITR, an AAV6 ITR, an AAV7 ITR, an AAV8 ITR, an AAVrh8 ITR, anAAV9 ITR, an AAV10 ITR, an AAVrh10 ITR, an AAV11 ITR, or an AAV12 ITR.In some embodiments, the AAV particle comprises an AAV vector encoding aheterologous transgene.

In some aspects, the invention provides a method to determine theserotype of an adeno-associated virus (AAV) particle comprising a)denaturing the AAV particle, b) subjecting the denatured AAV particle toreduction and/or alkylation, c) subjecting the denatured AAV particle todigestion to generate fragments of VP1, VP2 and/or VP3 of the AAVparticle, d) subjecting the fragments of VP1, VP2 and/or VP3 to liquidchromatography/mass spectrometry-mass spectrometry (LC/MS/MS), and e)determining the masses of fragments of VP1, VP2 and VP3 of the AAVparticle; wherein the specific combination of masses of fragments ofVP1, VP2 and VP3 are indicative of the AAV serotype. In someembodiments, the calculated masses of the fragments of VP1, VP2 and/orVP3 are compared to the theoretical masses of fragments of VP1, VP2and/or VP3 of one or more AAV serotypes.

In some aspects, the invention provides a method of determining theheterogeneity of a serotype of an AAV particle comprising a) denaturingthe AAV particle, b) subjecting the denatured AAV particle to reductionand/or alkylation, c) subjecting the denatured AAV particle to digestionto generate fragments of VP1, VP2 and/or VP3 of the AAV particle, d)subjecting the fragments of VP1, VP2 and/or VP3 to liquidchromatography/mass spectrometry-mass spectrometry (LC/MS/MS), e)determining the masses of fragments of VP1, VP2 and VP3 of the AAVparticle, and f) comparing the masses of step e) with the theoreticalmasses of fragments of VP1, VP2 and VP3 of the AAV serotype; wherein adeviation of one or more of the masses of VP1, VP2 or VP3 are indicativeof the AAV capsid heterogeneity. In some embodiments, the heterogeneitycomprises one or more of mixed serotypes, variant capsids, capsid aminoacid substitutions, truncated capsids, or modified capsids. In someembodiments, the reduction is by subjecting the AAV particle todithiothreitol, beta-mercaptoethanol, or tris(2-carboxyethyl)phosphine(TCEP). In some embodiments, the alkylation is by subjecting the AAVparticle to iodoacetic acid, iodoacetamide, or 4-vinylpyridine. In someembodiments, the digestion is an enzymatic digestion or a chemicaldigestion. In some embodiments, the enzymatic digestion is anendopeptidase digestion. In some embodiments, the enzymatic digestion isa trypsin digestion, a LysC digestion, an Asp-N digestion or a Glu-Cdigestion. In some embodiments, the chemical digestion is cyanogenbromide digestion or an acid digestion. In some embodiments, the AAVparticle is denatured with acetic acid, guanidine hydrochloride and/oran organic solvent.

In some embodiments of the above aspects and embodiments, the liquidchromatography is reverse phase liquid chromatography, size exclusionchromatography, hydrophilic interaction liquid chromatography, or cationexchange chromatography. In some embodiments, the liquid chromatographyis reverse phase liquid chromatography. In some embodiments, the reversephase chromatography is a C18 reverse chromatography. In someembodiments, the chromatography uses a mobile phase A comprising formicacid in water. In some embodiments, the mobile phase A comprises about0.1% formic acid. In some embodiments, the chromatography comprises amobile phase B comprising formic acid in acetonitrile. In someembodiments, the mobile phase B comprises about 0.1% formic acid. Insome embodiments, the proportion of mobile phase B in the chromatographyincreases over time. In some embodiments, mobile phase B increases fromabout 2% to about 60%. In some embodiments, mobile phase B increasesfrom about 2% to about 60% in about 121 minutes. In some embodiments,the liquid chromatography is high-performance liquid chromatography(HPLC).

In some embodiments of the above aspects and embodiments, the massspectrometry comprises a capillary voltage of about 3.5 kV. In someembodiments, the mass spectrometry comprises a sampling cone voltage ofabout 45 V. In some embodiments, the mass spectrometry comprisesassisted calibration. In some embodiments, sodium iodide is used as acalibrant.

In some embodiments of the above aspects and embodiments, the N-terminusof VP1 and/or VP3 is acetylated. In some embodiments, the AAV particleis a recombinant AAV (rAAV) particle. In some embodiments, the AAVparticle comprises an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, anAAV4 capsid, an AAV5 capsid, an AAV6 capsid, an AAV7 capsid, an AAV8capsid, an AAVrh8 capsid, an AAV9 capsid, an AAV10 capsid, an AAVrh10capsid, an AAV11 capsid, an AAV12 capsid, an AAV LK03 capsid, anAAV2R471A capsid, an AAV2/2-7m8 capsid, an AAV DJ capsid, an AAV DJ8capsid, an AAV2 N587A capsid, an AAV2 E548A capsid, an AAV2 N708Acapsid, an AAV V708K capsid, a goat AAV capsid, an AAV1/AAV2 chimericcapsid, a bovine AAV capsid, or a mouse AAV capsid rAAV2/HBoV1 (chimericAAV/human bocavirus virus 1). In some embodiments, the AAV capsidcomprises a tyrosine mutation or a heparin binding mutation. In someembodiments, the masses of VP1, VP2, and VP3 are compared to thetheoretical masses of one or more of AAV1 capsid, an AAV2 capsid, anAAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid, an AAV7capsid, an AAV8 capsid, an AAVrh8 capsid, an AAV9 capsid, an AAV10capsid, an AAVrh10 capsid, an AAV11 capsid, an AAV12 capsid, an AAV LK03capsid, an AAV2R471A capsid, an AAV2/2-7m8 capsid, an AAV DJ capsid, anAAV DJ8 capsid, an AAV2 N587A capsid, an AAV2 E548A capsid, an AAV2N708A capsid, an AAV V708K capsid, a goat AAV capsid, an AAV1/AAV2chimeric capsid, a bovine AAV capsid, or a mouse AAV capsid rAAV2/HBoV1(chimeric AAV/human bocavirus virus 1).

In some embodiments of the above aspects and embodiments, the viralparticle comprises an AAV1 ITR, an AAV2 ITR, an AAV3 ITR, an AAV4 ITR,an AAV5 ITR, an AAV6 ITR, an AAV7 ITR, an AAV8 ITR, an AAVrh8 ITR, anAAV9 ITR, an AAV10 ITR, an AAVrh10 ITR, an AAV11 ITR, or an AAV12 ITR.In some embodiments, the AAV particle comprises an AAV vector encoding aheterologous transgene.

In some embodiments, the invention provides a recombinant AAV (rAAV)particle comprising an amino acid substitution at amino acid residue 2of VP1 and/or VP3; wherein the amino acid substitution at amino acidresidue 2 of VP1 and/or VP3 alters N-terminal acetylation compared toN-terminal acetylation at amino acid residue 2 of VP1 and/or VP3 of theparent AAV particle. In some embodiments, the substitution results in ahigher frequency of N-terminal acetylation or a lower frequency ofN-terminal acetylation. In some embodiments, the rAAV particle comprisesan amino acid substitution at amino acid residue 2 of VP1; wherein theamino acid substitution at amino acid residue 2 of VP1 alters N-terminalacetylation compared to N-terminal acetylation at amino acid residue 2of VP1 of the parent AAV particle. In some embodiments, the rAAVparticle comprises an amino acid substitution at amino acid residue 2 ofVP3; wherein the amino acid substitution at amino acid residue 2 of VP3alters N-terminal acetylation compared to N-terminal acetylation atamino acid residue 2 of VP3 of the parent AAV particle. In someembodiments, amino acid residue 2 is substituted with Cys, Ser, Thr,Val, Gly, Asn, Asp, Glu, Ile, Leu, Phe, Gln, Lys, Met, Pro or Tyr. Insome embodiments, amino acid residue 2 is substituted with Ser, Asp orGlu.

In some embodiments of the above aspects and embodiments, the AAVparticle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV LK03,AAV2R471A, AAV2/2-7m8, AAV DJ, an AAV DJ8 capsid, AAV2 N587A, AAV2E548A, AAV2 N708A, AAV V708K, goat AAV, AAV1/AAV2 chimeric, bovine AAV,mouse AAV, rAAV2/HBoV1, AAV2HBKO, AAVPHP.B, or AAVPHP.eB serotypecapsid. In some embodiments, the AAV capsid further comprises a tyrosinemutation or a heparin binding mutation. In some embodiments, the rAAVparticle comprises a rAAV vector. In some embodiments, the rAAV vectorcomprises one or more AAV ITRs. In some embodiments, the rAAV vectorcomprises an AAV1 ITR, an AAV2 ITR, an AAV3 ITR, an AAV4 ITR, an AAV5ITR, an AAV6 ITR, an AAV7 ITR, an AAV8 ITR, an AAVrh8 ITR, an AAV9 ITR,an AAV10 ITR, an AAVrh10 ITR, an AAV11 ITR, or an AAV12 ITR. In someembodiments, the AAV capsid and the AAV ITRs are derived from the sameserotype. In some embodiments, the AAV capsid and the AAV ITRs arederived from different serotypes. In some embodiments, the AAV particlecomprises an AAV vector encoding a heterologous transgene flanked by oneor more AAV ITRs.

In some embodiments of the above aspects and embodiments, the rAAVvector is a self-complementary vector. In some embodiments, the rAAVvector comprises first nucleic acid sequence encoding the transgene anda second nucleic acid sequence encoding a complement of the transgene,wherein the first nucleic acid sequence can form intrastrand base pairswith the second nucleic acid sequence along most or all of its length.In some embodiments, the first nucleic acid sequence and the secondnucleic acid sequence are linked by a mutated AAV ITR, wherein themutated AAV ITR comprises a deletion of the D region and comprises amutation of the terminal resolution sequence.

In some embodiments of the above aspects and embodiments, the rAAVparticle is produced by transfecting a host cell with nucleic acidencoding the rAAV vector and nucleic acid encoding AAV rep and capfunctions, and providing nucleic acid encoding AAV helper functions. Insome embodiments, the AAV helper functions are provided by transfectingthe host cell with nucleic acid encoding the AAV helper functions. Insome embodiments, the AAV helper functions are provided by infecting thehost cell with an AAV helper virus that provides the AAV helperfunctions. In some embodiments, the AAV helper virus is an adenovirus, aherpes simplex virus or a baculovirus. In some embodiments, the rAAVparticle is produced by an AAV producer cell comprising nucleic acidencoding the rAAV vector and nucleic acid encoding AAV rep and capfunctions, and providing nucleic acid encoding AAV helper functions. Insome embodiments, the AAV producer cell comprises nucleic acid encodingAAV helper functions. In some embodiments, the AAV helper functions areprovided by infecting the AAV producer cells with an AAV helper virusthat provides the AAV helper functions. In some embodiments, the AAVhelper virus is an adenovirus, a herpes simplex virus, or a baculovirus.In some embodiments, the AAV cap functions provide an amino acidsubstitution at amino acid residue 2 of VP1 and/or VP3, wherein theamino acid substitution at amino acid residue 2 of VP1 and/or VP3 altersN-terminal acetylation compared to N-terminal acetylation at amino acidresidue 2 of VP1 and/or VP3 of the parent AAV particle.

In some aspects, the invention provides a pharmaceutical compositioncomprising the rAAV particle as described herein. In some aspects, theinvention provides a kit comprising the rAAV particle or thepharmaceutical composition as described herein. In some aspects, theinvention provides an article of manufacture comprising the rAAVparticle or the pharmaceutical composition as described herein.

In some aspects, the invention provides as AAV capsid protein comprisingan amino acid substitution at amino acid residue 2 of a parent AAVcapsid protein; wherein the amino acid substitution at amino acidresidue 2 alters N-terminal acetylation compared to N-terminalacetylation at amino acid residue 2 of the parent AAV capsid protein. Insome embodiments, the substitution results in a higher frequency ofN-terminal acetylation or a lower frequency of N-terminal acetylation.In some embodiments, the AAV capsid protein is VP1 or VP3. In someembodiments, amino acid residue 2 of the AAV capsid protein issubstituted with Cys, Ser, Thr, Val, Gly, Asn, Asp, Glu, Ile, Leu, Phe,Gln, Lys, Met, Pro or Tyr. In some embodiments, amino acid residue 2 ofthe AAV capsid protein is substituted with Ser, Asp or Glu. In someembodiments, the amino acid substitution results in less deamidation ofthe AAV capsid.

In some embodiments of the above aspects and embodiments, the AAVparticle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV LK03,AAV2R471A, AAV2/2-7m8, AAV DJ, an AAV DJ8 capsid, AAV2 N587A, AAV2E548A, AAV2 N708A, AAV V708K, goat AAV, AAV1/AAV2 chimeric, bovine AAV,mouse AAV, rAAV2/HBoV1, AAV2HBKO, AAVPHP.B, or AAVPHP.eB serotypecapsid. In some embodiments, the AAV capsid further comprises a tyrosinemutation or a heparin binding mutation.

In some aspects, the invention provides a method of improving stabilityof a rAAV particle comprising substituting amino acid residue 2 of VP1and/or VP3 of a parent VP1 and/or VP3; wherein the substituting aminoacid residue 2 alters N-terminal acetylation of VP1 and/or VP3, ascompared to amino acid residue 2 of the parent VP1 and/or VP3. In someaspects, the invention provides a method of improving assembly of rAAVparticles in a cell comprising substituting amino acid residue 2 of VP1and/or VP3 or a parental VP1 and/or VP3; wherein substituting amino acidat position 2 alters N-terminal acetylation of VP1 and/or VP3, ascompared to amino acid residue 2 of the parent VP1 and/or VP3. In someaspects, the invention provides a method of improving the transductionof rAAV particles in a cell comprising substituting amino acid residue 2of VP1 and/or VP3 or a parental VP1 and/or VP3; wherein substitutingamino acid residue 2 alters N-terminal acetylation of VP1 and/or VP3, ascompared to amino acid residue 2 of the parent VP1 and/or VP3. In someembodiments, the substituted amino acid results in a higher frequency ofN-terminal acetylation or a lower frequency of N-terminal acetylation.In some embodiments, the amino acid substitution at amino acid residue 2of VP1 is substituted. In some embodiments, the amino acid substitutionat amino acid residue 2 of VP3 is substituted. In some embodiments,amino acid residue 2 is substituted with Cys, Ser, Thr, Val, Gly, Asn,Asp, Glu, Ile, Leu, Phe, Gln, Lys, Met, Pro or Tyr. In some embodiments,amino acid residue 2 is substituted with Ser, Asp or Glu. In someaspects, the invention provides a method of reducing the transduction ofrAAV particles in a cell comprising substituting amino acid residue 2 ofVP1 and/or VP3; wherein the substituted amino acid at position 2 altersN-terminal acetylation of VP1 and/or VP3, as compared to amino acidresidue 2 of the parent VP1 and/or VP3.

In some embodiments of the above aspects and embodiments, the AAVparticle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV LK03,AAV2R471A, AAV2/2-7m8, AAV DJ, an AAV DJ8 capsid, AAV2 N587A, AAV2E548A, AAV2 N708A, AAV V708K, goat AAV, AAV1/AAV2 chimeric, bovine AAV,mouse AAV, rAAV2/HBoV1, AAV2HBKO, AAVPHP.B, or AAVPHP.eB serotypecapsid. In some embodiments, the AAV capsid further comprises a tyrosinemutation or a heparin binding mutation. In some embodiments, the rAAVparticle comprises a rAAV vector. In some embodiments, the rAAV vectorcomprises one or more AAV ITRs. In some embodiments, the rAAV vectorcomprises an AAV1 ITR, an AAV2 ITR, an AAV3 ITR, an AAV4 ITR, an AAV5ITR, an AAV6 ITR, an AAV7 ITR, an AAV8 ITR, an AAVrh8 ITR, an AAV9 ITR,an AAV10 ITR, an AAVrh10 ITR, an AAV11 ITR, or an AAV12 ITR. In someembodiments, the AAV capsid and the AAV ITRs are derived from the sameserotype. In some embodiments, the AAV capsid and the AAV ITRs arederived from different serotypes. In some embodiments, the AAV particlecomprises an AAV vector encoding a heterologous transgene flanked by oneor more AAV ITRs.

In some embodiments of the above aspects and embodiments, the rAAVvector is a self-complementary vector. In some embodiments, the rAAVvector comprises first nucleic acid sequence encoding the transgene anda second nucleic acid sequence encoding a complement of the transgene,wherein the first nucleic acid sequence can form intrastrand base pairswith the second nucleic acid sequence along most or all of its length.In some embodiments, the first nucleic acid sequence and the secondnucleic acid sequence are linked by a mutated AAV ITR, wherein themutated AAV ITR comprises a deletion of the D region and comprises amutation of the terminal resolution sequence.

In some embodiments of the above aspects and embodiments, the rAAVparticle is produced by transfecting a host cell with nucleic acidencoding the rAAV vector and nucleic acid encoding AAV rep and capfunctions, and providing nucleic acid encoding AAV helper functions. Insome embodiments, the AAV helper functions are provided by transfectingthe host cell with nucleic acid encoding the AAV helper functions. Insome embodiments, the AAV helper functions are provided by infecting thehost cell with an AAV helper virus that provides the AAV helperfunctions. In some embodiments, the AAV helper virus is an adenovirus, aherpes simplex virus or a baculovirus. In some embodiments, the rAAVparticle is produced by an AAV producer cell comprising nucleic acidencoding the rAAV vector and nucleic acid encoding AAV rep and capfunctions, and providing nucleic acid encoding AAV helper functions. Insome embodiments, the AAV producer cell comprises nucleic acid encodingAAV helper functions. In some embodiments, the AAV helper functions areprovided by infecting the AAV producer cells with an AAV helper virusthat provides the AAV helper functions. In some embodiments, the AAVhelper virus is an adenovirus, a herpes simplex virus, or a baculovirus.In some embodiments, the AAV cap functions provide an amino acidsubstitution at amino acid residue 2 of VP1 and/or VP3, wherein theamino acid substitution at amino acid residue 2 of VP1 and/or VP3 altersN-terminal acetylation compared to N-terminal acetylation at amino acidresidue 2 of VP1 and/or VP3 of the parent AAV particle.

In some aspects, the invention provides a recombinant AAV (rAAV)particle comprising one or more amino acid substitutions at amino acidresidue A35, N57, G58, N382, G383, N511, G512, N715, or G716 of VP1 orVP3 of a parent particle, residue numbering based on VP1 of AAV2;wherein the one or more amino acid substitutions alters deamidation ascompared to deamidation of VP1 and/or VP3 of the parent AAV particle. Insome embodiments, the one or more amino acid substitution is at aminoacid residue A35, N57 of VP1, G58 of VP1, N382 of VP3, G383 of VP3, N511of VP3, G512 of VP3, N715 of VP3, or G716 of VP3 and alters deamidationas compared to deamidation of VP1 and/or VP3 of the parent AAV particle.In some embodiments, the one or more amino acid substitutions comprisesa substitution with Asp at N57 of VP1, N382 of VP3, N511 of VP3, or N715of VP3; and results in a higher frequency of deamidation as compared todeamidation of VP1 and/or VP3 of the parent AAV particle. In someembodiments, the one or more amino acid substitutions comprise a N57K ora N57Q substitution and results in a lower frequency of deamidation ascompared to deamidation of VP1 and/or VP3 of the parent AAV particle. Insome embodiments, the one or more amino acid substitution comprise asubstitution with Asp at A35 of VP1 and results in a higher frequency ofdeamidation as compared to deamidation of VP1 of the parent AAVparticle. In some embodiments, the one or more amino acid substitutionsis at G58 of VP1, G383 of VP3, G512 of VP3, or G716 of VP3 and resultsin a lower frequency of deamidation as compared to deamidation of VP1and/or VP3 of the parent AAV particle. In some embodiments, the G58 ofVP1 is substituted with Asp. In some embodiments, the rAAV particle isan AAV1 particle or an AAV2 particle.

In some aspects, the invention provides pharmaceutical compositionscomprising AAV particles comprising one or more amino acid substitutionsat amino acid residue A35, N57, G58, N382, G383, N511, G512, N715, orG716 of VP1 or VP3, residue numbering based on VP1 of AAV2; wherein theamino acid substitution alters deamidation as compared to deamidation ofVP1 and/or VP3 of the parent AAV particle. In some aspects, theinvention provides kits comprising AAV particles or compositionscomprising AAV particles wherein the AAV particles comprise one or moreamino acid substitutions at amino acid residue A35, N57, G58, N382,G383, N511, G512, N715, or G716 of VP1 or VP3, residue numbering basedon VP1 of AAV2; wherein the amino acid substitution alters deamidationas compared to deamidation of VP1 and/or VP3 of the parent AAV particle.In some aspects, the invention provides articles of manufacturecomprising AAV particles or compositions comprising AAV particleswherein the AAV particles comprise one or more amino acid substitutionsat amino acid residue A35, N57, G58, N382, G383, N511, G512, N715, orG716 of VP1 or VP3, residue numbering based on VP1 of AAV2; wherein theamino acid substitution alters deamidation as compared to deamidation ofVP1 and/or VP3 of the parent AAV particle. In some aspects, theinvention provides an AAV capsid protein comprising an amino acidsubstitution of a parent AAV capsid protein; wherein the amino acidsubstitution alters deamidation of the capsid compared to the parent AAVcapsid protein.

In some aspects, the invention provides a method of improving thestability of a rAAV particle comprising substituting one or more aminoacid residues, wherein the one or more amino acid residues is A35, N57,G58, N382, G383, N511, G512, N715, or G716, residue numbering based onVP1 of AAV2; wherein the amino acid substitution alters deamidation ascompared to deamidation of VP1 and/or VP3 of the parent AAV particle. Insome aspects, the invention provides a method of improving the assemblyof rAAV particles in a cell comprising substituting one or more aminoacid residues, wherein the one or more amino acid residues is A35, N57,G58, N382, G383, N511, G512, N715, or G716, residue numbering based onVP1 of AAV2; wherein the amino acid substitution alters deamidation ascompared to deamidation of VP1 and/or VP3 of the parent AAV particle. Insome aspects, the invention provides a method of improving thetransduction of rAAV particles in a cell comprising substituting one ormore amino acid residues, wherein the one or more amino acid residues isA35, N57, G58, N382, G383, N511, G512, N715, or G716, residue numberingbased on VP1 of AAV2; wherein the amino acid substitution altersdeamidation as compared to deamidation of VP1 and/or VP3 of the parentAAV particle. In some embodiments, the one or more amino acidsubstitutions is at A35 of VP1, N57 of VP1, G58 of VP1, N382 of VP3,G383 of VP3, N511 of VP3, G512 of VP3, N715 of VP3, or G716 of VP3;wherein the amino acid substitution alters deamidation as compared todeamidation of VP1 and/or VP3 of the parent AAV particle. In someembodiments, the parental Ala residue at position 35 of VP1 issubstituted with Asn. In some embodiments, the parental Gly residue atposition 58 of VP1 is substituted with Asp. In some embodiments, therAAV particle is an AAV1 particle or an AAV2 particle.

In some embodiments, the invention provides a method of improving thestability, assembly and/or transduction efficiency of a rAAV particlecomprising substituting one or more amino acid residues, wherein the oneor more amino acid residues is A35, N57, G58, N382, G383, N511, G512,N715, or G716, residue numbering based on VP1 of AAV2; wherein the aminoacid substitution alters deamidation as compared to deamidation of VP1and/or VP3 of the parent AAV particle as described above, wherein theAAV particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV LK03,AAV2R471A, AAV2/2-7m8, AAV DJ, an AAV DJ8 capsid, AAV2 N587A, AAV2E548A, AAV2 N708A, AAV V708K, goat AAV, AAV1/AAV2 chimeric, bovine AAV,mouse AAV, or rAAV2/HBoV1 serotype capsid. In some embodiments, the AAVcapsid further comprises a tyrosine mutation or a heparin bindingmutation. In some embodiments, the rAAV particle comprises a rAAVvector. In some embodiments, the rAAV vector comprises one or more AAVITRs. In some embodiments, the rAAV vector comprises an AAV1 ITR, anAAV2 ITR, an AAV3 ITR, an AAV4 ITR, an AAV5 ITR, an AAV6 ITR, an AAV7ITR, an AAV8 ITR, an AAVrh8 ITR, an AAV9 ITR, an AAV10 ITR, an AAVrh10ITR, an AAV11 ITR, or an AAV12 ITR. In some embodiments, the AAV capsidand the AAV ITRs are derived from the same serotype. In someembodiments, the AAV capsid and the AAV ITRs are derived from differentserotypes. In some embodiments, the AAV particle comprises an AAV vectorencoding a heterologous transgene flanked by one or more AAV ITRs.

In some embodiments of the above aspects and embodiments, the rAAVvector is a self-complementary vector. In some embodiments, the rAAVvector comprises first nucleic acid sequence encoding the transgene anda second nucleic acid sequence encoding a complement of the transgene,wherein the first nucleic acid sequence can form intrastrand base pairswith the second nucleic acid sequence along most or all of its length.In some embodiments, the first nucleic acid sequence and the secondnucleic acid sequence are linked by a mutated AAV ITR, wherein themutated AAV ITR comprises a deletion of the D region and comprises amutation of the terminal resolution sequence.

In some embodiments of the above aspects and embodiments, the rAAVparticle is produced by transfecting a host cell with nucleic acidencoding the rAAV vector and nucleic acid encoding AAV rep and capfunctions, and providing nucleic acid encoding AAV helper functions. Insome embodiments, the AAV helper functions are provided by transfectingthe host cell with nucleic acid encoding the AAV helper functions. Insome embodiments, the AAV helper functions are provided by infecting thehost cell with an AAV helper virus that provides the AAV helperfunctions. In some embodiments, the AAV helper virus is an adenovirus, aherpes simplex virus or a baculovirus. In some embodiments, the rAAVparticle is produced by an AAV producer cell comprising nucleic acidencoding the rAAV vector and nucleic acid encoding AAV rep and capfunctions, and providing nucleic acid encoding AAV helper functions. Insome embodiments, the AAV producer cell comprises nucleic acid encodingAAV helper functions. In some embodiments, the AAV helper functions areprovided by infecting the AAV producer cells with an AAV helper virusthat provides the AAV helper functions. In some embodiments, the AAVhelper virus is an adenovirus, a herpes simplex virus, or a baculovirus.In some embodiments, the AAV cap functions provide an amino acidsubstitution of VP1 and/or VP3, wherein the amino acid substitutionmodulated deamidation of the capsid compared to the parent AAV particle.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D provide total ion Chromatograms of LC/MS of AAV2 VPs. FIG.1A: 10 cm long BEH C4 column with 1.7%/min gradient, FIG. 1B: 10 cm longBEH C4 column with 0.5%/min gradient; FIG. 1C: 15 cm long BEH C4 columnwith 0.5%/min gradient, FIG. 1D: 15 cm long BEH C8 column with 0.5%/mingradient.

FIGS. 2A&B provide deconvoluted mass spectra from FIG. 1D peak 1 (FIG.2A) and FIG. 1D peak 2 (FIG. 2B).

FIG. 3 provides the sequence coverage of AAV2 VP1 (SEQ ID NO:3): green,tryptic peptides, blue, Lys-C peptides, pink, Asp-N peptides.

FIGS. 4A-4C provide MS/MS spectra of AAV2 VP N-terminal peptides. FIG.4A: VP1 N-terminal tryptic peptide A(Ac)ADGYLPDWLEDTLSEGIR (SEQ ID NO:4), FIG. 4B VP2 N-terminal Asp-N peptide APGKKRPVEHSPVEP (SEQ ID NO:15). FIG. 4C: VP-3 N-terminal Asp-N derived peptide A(Ac)TGSGAPM (SEQ IDNO: 5).

FIG. 5 provides the sequence alignment of 13 AAV serotypes blackletter/white background: non-similar; blue letter/blue background:conservative; black letter/green background: block of similar; redletter/yellow background: identical; green letter/white background:weakly similar. AAVRh10 (SEQ ID NO: 17); AAV10 (SEQ ID NO: 18); AAV8(SEQ ID NO: 19); AAV7 (SEQ ID NO: 20); AAV1 (SEQ ID NO: 21); AAV6 (SEQID NO: 22); AAV2 (SEQ ID NO: 23); AAV3 (SEQ ID NO: 24); AAV11 (SEQ IDNO: 25); AAV12 (SEQ ID NO: 26); AAV4 (SEQ ID NO: 27); AAV5 (SEQ ID NO:28); AAV9 (SEQ ID NO: 29); Consensus (SEQ ID NO: 30).

FIGS. 6A & 6B show the results of LC/MS/MS analysis comparing thepercentage of deamidation in AAV1 and AAV2 particles produced by the TTxand PCL methods. The T9 peptide YLGPFNGLDK (SEQ ID NO: 9) was used tomonitor potential deamidation site N57 in both AAV1 and AAV2.

FIGS. 7A & 7B show the results of LC/MS/MS analysis comparing thepercentage of deamidation in AAV1 and AAV2 particles produced by the TTxand PCL methods. The T49 peptides YNLNGR (SEQ ID NO: 11) and YHLNGR (SEQID NO: 12) were used to monitor potential deamidation site N511 in AAV1and AAV2, respectively.

FIGS. 8A & 8B show the results of LC/MS/MS analysis comparing thepercentage of deamidation in AAV1 and AAV2 particles produced by the TTxand PCL methods. The T67 peptides SANVDFTVDNNGLYTEPR (SEQ ID NO: 13) andSVNVDFTVDTNGVYSEPR (SEQ ID NO: 14) were used to monitor potentialdeamidation site N715 in AAV1 and AAV2, respectively.

FIG. 9 shows the results of SYPRO protein gel analysis of production andVP1:VP2:VP3 ratio of AAV5 deacetylated mutant variants.

FIG. 10 illustrates an in vitro transduction assay for testingtransduction efficiency of AAV5 deacetylated variants.

FIG. 11 shows the efficiency of cell entry by the indicated AAV5deacetylated variants or parental unmodified AAV5, as measured by vectorgenome copies/μg protein. Three cell lines were used: 293, HeLa, andHuH7.

FIG. 12 shows eGFP expression (as measured by ELISA) by cells transducedwith the indicated AAV5 deacetylated variants as compared totransduction with parental unmodified AAV5. Three cell lines were used:293, HeLa, and HuH7.

FIG. 13 provides the sequence alignment of 13 AAV serotypes,highlighting the conserved N57G58 deamidation site and the A35 residuein AAV2. AAVRh10 (SEQ ID NO: 31); AAV10 (SEQ ID NO: 31); AAV8 (SEQ IDNO: 32); AAV7 (SEQ ID NO: 33); AAV1 (SEQ ID NO: 31); AAV6 (SEQ ID NO:31); AAV2 (SEQ ID NO: 34); AAV3 (SEQ ID NO: 35); AAV11 (SEQ ID NO: 31);AAV12 (SEQ ID NO: 36); AAV4 (SEQ ID NO: 37); AAV5 (SEQ ID NO: 38); AAV9(SEQ ID NO: 39); Consensus (SEQ ID NO: 40).

FIG. 14 shows a protein gel of VP1, VP2, and VP3 capsid proteins fromAAV1 or AAV2 particles produced by the PCL or TTx method. *highlightsthe truncated VP1 (tVP1) protein.

FIG. 15 shows the results of LC/MS analysis of deamidation of theindicated AAV2 mutants, as compared to control AAV2 capsids.

FIG. 16 shows the results of SYPRO protein gel analysis of productionand VP1:VP2:VP3 ratio of AAV2 deamidation mutant variants.

FIG. 17 illustrates an in vitro transduction assay for testingtransduction efficiency of AAV2 deamidation variants.

FIG. 18 shows the efficiency of cell entry by the indicated AAV2deamidation variants or parental unmodified AAV2, as measured by vectorgenome copies/μg protein. Three cell lines were used: 293, HeLa, andHuH7.

FIG. 19 shows eGFP expression (as measured by ELISA) by cells transducedwith the indicated AAV2 deamidation variants as compared to transductionwith parental unmodified AAV2. Three cell lines were used: 293, HeLa,and HuH7.

DETAILED DESCRIPTION

In some aspects, the invention provides a method to determine theserotype of an adeno-associated virus (AAV) particle(s) comprising: a)denaturing the AAV particle, b) injecting the denatured AAV particle toliquid chromatography/mass spectrometry (LC/MS), and c) determining themasses of VP1, VP2 and VP3 of the AAV particle; wherein the specificcombination of masses of VP1, VP2 and VP3 are indicative of the AAVserotype.

In other aspects, the invention provides a method of determining theheterogeneity of an AAV particle comprising: a) denaturing the AAVparticle, b) injecting the denatured AAV particle to liquidchromatography/mass spectrometry (LC/MS), and c) determining the massesof VP1, VP2 and VP3 of the AAV particle, and comparing the masses ofstep c) with the theoretical masses of VP1, VP2 and VP3 of the AAVserotype; wherein a deviation of one or more of the masses of VP1, VP2or VP3 are indicative of the AAV capsid heterogeneity.

In other aspects, the invention provides a method to determine theserotype of an adeno-associated virus (AAV) particle comprising a)denaturing the AAV particle, b) subjecting the denatured AAV particle toreduction and/or alkylation, c) injecting the denatured AAV particle todigestion to generate fragments of VP1, VP2 and/or VP3 of the AAVparticle, d) subjecting the fragments of VP1, VP2 and/or VP3 to liquidchromatography/mass spectrometry-mass spectrometry (LC/MS/MS), and e)determining the masses of fragments of VP1, VP2 and VP3 of the AAVparticle; wherein the specific combination of masses of fragments ofVP1, VP2 and VP3 are indicative of the AAV serotype.

In other aspects, the invention provides a method of determining theheterogeneity of an AAV particle of a serotype comprising: a) denaturingthe AAV particle, b) subjecting the denatured AAV particle to reductionand/or alkylation, c) injecting the denatured AAV particle to digestionto generate fragments of VP1, VP2 and/or VP3 of the AAV particle, d)subjecting the fragments of VP1, VP2 and/or VP3 to liquidchromatography/mass spectrometry-mass spectrometry (LC/MS/MS), e)determining the masses of fragments of VP1, VP2 and VP3 of the AAVparticle, and f) comparing the masses of step e) with the theoreticalmasses of fragments of VP1, VP2 and VP3 of the AAV serotype; wherein adeviation of one or more of the masses of VP1, VP2 or VP3 are indicativeof the AAV capsid heterogeneity.

In some aspects, the invention provides a recombinant AAV (rAAV)particle comprising an amino acid substitution at amino acid residue 2of VP1 and/or VP3; wherein the amino acid substitution at amino acidresidue 2 of VP1 and/or VP3 alters N-terminal acetylation compared toN-terminal acetylation at amino acid residue 2 of VP1 and/or VP3 of theparent AAV particle.

In some aspects, the invention provides a method of improving theassembly of rAAV particles in a cell comprising substituting amino acidresidue 2 of VP1 and/or VP3; wherein the substituted amino acid atposition 2 is N-acetlylated at a higher frequency than amino acidresidue 2 of the parent VP1 and/or VP3. In some aspects, the inventionprovides a method of improving the transduction of rAAV particles in acell comprising substituting amino acid residue 2 of VP1 and/or VP3;wherein the substituted amino acid at position 2 is N-acetylated at ahigher frequency than amino acid residue 2 of the parent VP1 and/or VP3.

I. General Techniques

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Molecular Cloning: ALaboratory Manual (Sambrook et al., 4^(th) ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 2012); Current Protocols inMolecular Biology (F. M. Ausubel, et al. eds., 2003); the series Methodsin Enzymology (Academic Press, Inc.); PCR 2: A Practical Approach (M. J.MacPherson, B. D. Hames and G. R. Taylor eds., 1995); Antibodies, ALaboratory Manual (Harlow and Lane, eds., 1988); Culture of AnimalCells: A Manual of Basic Technique and Specialized Applications (R. I.Freshney, 6^(th) ed., J. Wiley and Sons, 2010); OligonucleotideSynthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, HumanaPress; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., AcademicPress, 1998); Introduction to Cell and Tissue Culture (J. P. Mather andP. E. Roberts, Plenum Press, 1998); Cell and Tissue Culture: LaboratoryProcedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., J. Wileyand Sons, 1993-8); Handbook of Experimental Immunology (D. M. Weir andC. C. Blackwell, eds., 1996); Gene Transfer Vectors for Mammalian Cells(J. M. Miller and M. P. Calos, eds., 1987); PCR: The Polymerase ChainReaction, (Mullis et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology(Ausubel et al., eds., J. Wiley and Sons, 2002); Immunobiology (C. A.Janeway et al., 2004); Antibodies (P. Finch, 1997); Antibodies: APractical Approach (D. Catty., ed., IRL Press, 1988-1989); MonoclonalAntibodies: A Practical Approach (P. Shepherd and C. Dean, eds., OxfordUniversity Press, 2000); Using Antibodies: A Laboratory Manual (E.Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); TheAntibodies (M. Zanetti and J. D. Capra, eds., Harwood AcademicPublishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., eds., J.B. Lippincott Company, 2011).

II. Definitions

A “vector,” as used herein, refers to a recombinant plasmid or virusthat comprises a nucleic acid to be delivered into a host cell, eitherin vitro or in vivo.

The term “polynucleotide” or “nucleic acid” as used herein refers to apolymeric form of nucleotides of any length, either ribonucleotides ordeoxyribonucleotides. Thus, this term includes, but is not limited to,single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA,DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases, orother natural, chemically or biochemically modified, non-natural, orderivatized nucleotide bases. The backbone of the nucleic acid cancomprise sugars and phosphate groups (as may typically be found in RNAor DNA), or modified or substituted sugar or phosphate groups.Alternatively, the backbone of the nucleic acid can comprise a polymerof synthetic subunits such as phosphoramidates and thus can be anoligodeoxynucleoside phosphoramidate (P—NH₂) or a mixedphosphoramidate-phosphodiester oligomer. In addition, a double-strandednucleic acid can be obtained from the single stranded polynucleotideproduct of chemical synthesis either by synthesizing the complementarystrand and annealing the strands under appropriate conditions, or bysynthesizing the complementary strand de novo using a DNA polymerasewith an appropriate primer.

The terms “polypeptide” and “protein” are used interchangeably to referto a polymer of amino acid residues, and are not limited to a minimumlength. Such polymers of amino acid residues may contain natural ornon-natural amino acid residues, and include, but are not limited to,peptides, oligopeptides, dimers, trimers, and multimers of amino acidresidues. Both full-length proteins and fragments thereof areencompassed by the definition. The terms also include post-translationalmodifications of the polypeptide, for example, glycosylation,sialylation, acetylation, phosphorylation, and the like. Furthermore,for purposes of the present invention, a “polypeptide” refers to aprotein which includes modifications, such as deletions, additions, andsubstitutions (generally conservative in nature), to the nativesequence, as long as the protein maintains the desired activity. Thesemodifications may be deliberate, as through site-directed mutagenesis,or may be accidental, such as through mutations of hosts which producethe proteins or errors due to PCR amplification.

A “recombinant viral vector” refers to a recombinant polynucleotidevector comprising one or more heterologous sequences (i.e., nucleic acidsequence not of viral origin). In the case of recombinant AAV vectors,the recombinant nucleic acid is flanked by at least one, e.g., two,inverted terminal repeat sequences (ITRs).

A “recombinant AAV vector (rAAV vector)” refers to a polynucleotidevector comprising one or more heterologous sequences (i.e., nucleic acidsequence not of AAV origin) that are flanked by at least one, e.g., two,AAV inverted terminal repeat sequences (ITRs). Such rAAV vectors can bereplicated and packaged into infectious viral particles when present ina host cell that has been infected with a suitable helper virus (or thatis expressing suitable helper functions) and that is expressing AAV repand cap gene products (i.e. AAV Rep and Cap proteins). When a rAAVvector is incorporated into a larger polynucleotide (e.g., in achromosome or in another vector such as a plasmid used for cloning ortransfection), then the rAAV vector may be referred to as a “pro-vector”which can be “rescued” by replication and encapsidation in the presenceof AAV packaging functions and suitable helper functions. A rAAV vectorcan be in any of a number of forms, including, but not limited to,plasmids, linear artificial chromosomes, complexed with lipids,encapsulated within liposomes, and, in embodiments, encapsidated in aviral particle, particularly an AAV particle. A rAAV vector can bepackaged into an AAV virus capsid to generate a “recombinantadeno-associated viral particle (rAAV particle)”.

An “rAAV virus” or “rAAV viral particle” refers to a viral particlecomposed of at least one AAV capsid protein and an encapsidated rAAVvector genome.

A “parent AAV particle” and “parent AAV capsid protein” as used hereinin the context of comparing N-acetylation and/or deamidation refers toan AAV particle or capsid protein into which amino acid modificationsare introduced to modulate N-acetylation and/or deamidation (e.g., anAAV particle/capsid protein that is the same as or similar to the AAVparticle/capsid of the subject invention but does not comprise themutations that modulate/alter N-acetylation and/or deamidation asdescribed herein). In some embodiments, the parent AAV particle is arecombinant AAV particle comprising a recombinant AAV genome. In someembodiments, the parent AAV capsid particle or parent AAV capsid proteincomprises amino acid substitutions that affect other aspects of the AAVparticle. For example, the parent AAV particle may comprise amino acidsubstitutions that affect the binding of AAV to its receptor, such asaffecting binding of AAV2 to heparin sulfate proteoglycan (e.g. an AAV2HBKO particle). An AAV2 HBKO particle can be mutated to introduce aminoacid substitutions that modulate N-acetylation and/or deamidation. Sucha mutated AAV particle may then be compared to the parent AAV2 HBKOparticle in aspects of the invention as described herein. A parent AAVcapsid protein may include a parent VP1 capsid protein, a parent VP2capsid protein, or a VP3 capsid protein.

As used herein, the term “modulate” or “alter” in reference to a parentmolecule means to change a feature of the parent molecule. For example,an AAV particle with altered N-acetlylation may show increased ordecreased N-acetylation compared to the parent AAV particle and an AAVparticle with altered deamidation may show increased or decreaseddeamidation compared to the parent AAV particle.

“Heterologous” means derived from a genotypically distinct entity fromthat of the rest of the entity to which it is compared or into which itis introduced or incorporated. For example, a nucleic acid introduced bygenetic engineering techniques into a different cell type is aheterologous nucleic acid (and, when expressed, can encode aheterologous polypeptide). Similarly, a cellular sequence (e.g., a geneor portion thereof) that is incorporated into a viral vector is aheterologous nucleotide sequence with respect to the vector.

The term “transgene” refers to a nucleic acid that is introduced into acell and is capable of being transcribed into RNA and optionally,translated and/or expressed under appropriate conditions. In aspects, itconfers a desired property to a cell into which it was introduced, orotherwise leads to a desired therapeutic or diagnostic outcome. Inanother aspect, it may be transcribed into a molecule that mediates RNAinterference, such as siRNA.

The terms “genome particles (gp),” “genome equivalents,” or “genomecopies” as used in reference to a viral titer, refer to the number ofvirions containing the recombinant AAV DNA genome, regardless ofinfectivity or functionality. The number of genome particles in aparticular vector preparation can be measured by procedures such asdescribed in the Examples herein, or for example, in Clark et al. (1999)Hum. Gene Ther., 10:1031-1039; Veldwijk et al. (2002) Mol. Ther.,6:272-278.

The terms “infection unit (iu),” “infectious particle,” or “replicationunit,” as used in reference to a viral titer, refer to the number ofinfectious and replication-competent recombinant AAV vector particles asmeasured by the infectious center assay, also known as replicationcenter assay, as described, for example, in McLaughlin et al. (1988) J.Virol., 62:1963-1973.

The term “transducing unit (tu)” as used in reference to a viral titer,refers to the number of infectious recombinant AAV vector particles thatresult in the production of a functional transgene product as measuredin functional assays such as described in Examples herein, or forexample, in Xiao et al. (1997) Exp. Neurobiol., 144:113-124; or inFisher et al. (1996) J. Virol., 70:520-532 (LFU assay).

An “inverted terminal repeat” or “ITR” sequence is a term wellunderstood in the art and refers to relatively short sequences found atthe termini of viral genomes which are in opposite orientation.

An “AAV inverted terminal repeat (ITR)” sequence, a term well-understoodin the art, is an approximately 145-nucleotide sequence that is presentat both termini of the native single-stranded AAV genome. The outermost125 nucleotides of the ITR can be present in either of two alternativeorientations, leading to heterogeneity between different AAV genomes andbetween the two ends of a single AAV genome. The outermost 125nucleotides also contains several shorter regions ofself-complementarity (designated A, A′, B, B′, C, C′ and D regions),allowing intrastrand base-pairing to occur within this portion of theITR.

A “terminal resolution sequence” or “trs” is a sequence in the D regionof the AAV ITR that is cleaved by AAV rep proteins during viral DNAreplication. A mutant terminal resolution sequence is refractory tocleavage by AAV rep proteins. “AAV helper functions” refer to functionsthat allow AAV to be replicated and packaged by a host cell. AAV helperfunctions can be provided in any of a number of forms, including, butnot limited to, helper virus or helper virus genes which aid in AAVreplication and packaging. Other AAV helper functions are known in theart such as genotoxic agents.

“AAV helper functions” refer to functions that allow AAV to bereplicated and packaged by a host cell. AAV helper functions can beprovided in any of a number of forms, including, but not limited to,helper virus or helper virus genes which aid in AAV replication andpackaging. Other AAV helper functions are known in the art such asgenotoxic agents.

A “helper virus” for AAV refers to a virus that allows AAV (which is adefective parvovirus) to be replicated and packaged by a host cell. Anumber of such helper viruses have been identified, includingadenoviruses, herpesviruses, poxviruses such as vaccinia, andbaculovirus. The adenoviruses encompass a number of different subgroups,although Adenovirus type 5 of subgroup C (Ad5) is most commonly used.Numerous adenoviruses of human, non-human mammalian and avian origin areknown and are available from depositories such as the ATCC. Viruses ofthe herpes family, which are also available from depositories such asATCC, include, for example, herpes simplex viruses (HSV), Epstein-Barrviruses (EBV), cytomegaloviruses (CMV) and pseudorabies viruses (PRV).Baculoviruses available from depositories include Autographa californicanuclear polyhedrosis virus.

“Percent (%) sequence identity” with respect to a reference polypeptideor nucleic acid sequence is defined as the percentage of amino acidresidues or nucleotides in a candidate sequence that are identical withthe amino acid residues or nucleotides in the reference polypeptide ornucleic acid sequence, after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity,and not considering any conservative substitutions as part of thesequence identity. Alignment for purposes of determining percent aminoacid or nucleic acid sequence identity can be achieved in various waysthat are within the skill in the art, for instance, using publiclyavailable computer software programs, for example, those described inCurrent Protocols in Molecular Biology (Ausubel et al., eds., 1987),Supp. 30, section 7.7.18, Table 7.7.1, and including BLAST, BLAST-2,ALIGN or Megalign (DNASTAR) software. A potential alignment program isALIGN Plus (Scientific and Educational Software, Pennsylvania). Thoseskilled in the art can determine appropriate parameters for measuringalignment, including any algorithms needed to achieve maximal alignmentover the full length of the sequences being compared. For purposesherein, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows: 100 times thefraction X/Y, where X is the number of amino acid residues scored asidentical matches by the sequence alignment program in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A. For purposes herein, the % nucleic acidsequence identity of a given nucleic acid sequence C to, with, oragainst a given nucleic acid sequence D (which can alternatively bephrased as a given nucleic acid sequence C that has or comprises acertain % nucleic acid sequence identity to, with, or against a givennucleic acid sequence D) is calculated as follows: 100 times thefraction W/Z, where W is the number of nucleotides scored as identicalmatches by the sequence alignment program in that program's alignment ofC and D, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C.

An “isolated” molecule (e.g., nucleic acid or protein) or cell means ithas been identified and separated and/or recovered from a component ofits natural environment.

“Mass spectrometry” refers to the analytical chemistry technique ofidentifying an amount and/or type of a compound (e.g., a polypeptide) bymeasuring the mass-to-charge ratio and abundance of gas-phase ions. Theterm “mass spectrometry” may be used interchangeably herein.

“Heterogeneity” when used in reference to an AAV capsid refers to an AAVcapsid characterized by one or more capsid polypeptides observed todeviate from a reference mass of a VP1, VP2, and/or VP3 polypeptide, orfragment thereof. A reference mass may include, without limitation, atheoretical, predicted, or expected mass of a VP1, VP2, and/or VP3polypeptide, e.g., of a known AAV serotype. For example, an AAV capsidmay be said to display heterogeneity if it demonstrates one or more ofthe following properties (without limitation): a mixed serotype, avariant capsid, a capsid amino acid substitution, a truncated capsid, ora modified capsid.

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X.”

As used herein, the singular form of the articles “a,” “an,” and “the”includes plural references unless indicated otherwise.

It is understood that aspects and embodiments of the invention describedherein include “comprising,” “consisting,” and/or “consistingessentially of” aspects and embodiments.

III. Methods

Certain aspects of the present disclosure relate to methods ofdetermining the serotype of a viral particle. Other aspects of thepresent disclosure relate to methods of determining the heterogeneity ofa viral particle. As described below, the accurate masses of VP1, VP2and VP3 of each AAV serotype are unique and can be used to identify ordifferentiate AAV capsid serotypes. These methods are based in part onthe discovery described herein that direct LC/MS of different types ofAAVs after denaturation may be used to monitor the protein sequence andpost-translational modifications with accurate mass measurement in theintact protein level. Further, acetylations of N-termini of VP1 and VP3may also be identified and/or monitored in different AAV serotypes.Based on these AAV results and the guidance provided herein, it iscontemplated that such methods may readily be applied to profile avariety of viruses, e.g., the viral families, subfamilies, and genera ofthe present disclosure. The methods of the present disclosure may finduse, e.g., in profile VPs to monitor VP expressions, posttranslationalmodifications, and truncations and to ensure product consistency duringVLP production, to confirm site-direct mutagenesis or structuralcharacterization for capsid protein engineering applications, and/or tomonitor or detect heterogeneity of a viral particle or preparation.

In some embodiments, the methods include denaturing a viral particle. Insome embodiments, a viral particle such as an AAV particle may bedenatured using detergent, heat, high salt, or buffering with a low orhigh pH. In certain embodiments, an AAV particle may be denatured usingacetic acid or guanidine hydrochloride. The skilled artisan willrecognize that a variety of methods useful for promoting and/ormonitoring protein denaturation are available in the art and maysuitably select a denaturation method compatible with liquidchromatography/mass spectrometry. For example, if heat denaturation isused, care may be applied to avoid protein precipitation and reversephase column clogging. Similarly, high salt denaturation may be coupledwith a desalting step prior to LC/MS or LC/MS/MS. In other embodiments,high pH denaturation, low pH denaturation, or denaturation using organicsolvents is used.

In some embodiments, the methods include subjecting a denatured viralparticle of the present disclosure to liquid chromatography/massspectrometry (LC/MS). As is known in the art, LC/MS utilizes liquidchromatography for physical separation of ions and mass spectrometry forgeneration of mass spectral data from the ions. Such mass spectral datamay be used to determine, e.g., molecular weight or structure,identification of particles by mass, quantity, purity, and so forth.These data may represent properties of the detected ions such as signalstrength (e.g., abundance) over time (e.g., retention time), or relativeabundance over mass-to-charge ratio.

In some embodiments, liquid chromatography (e.g., used in LC/MS asdescribed herein) is ultra-performance liquid chromatography (UPLC; theterm “ultra high performance liquid chromatography” or UHPLC may be usedinterchangeably herein). UPLC is known in the art as an LC techniquethat relies upon a column with reduced particle size (e.g., less than 2μm) and increased flow velocity to improve chromatographic resolution,efficiency, peak capacity, and sensitivity (see, e.g., Plumb, R. et al.(2004) Rapid Commun. Mass Spectrom. 18:2331-2337). In some embodiments,UPLC refers to the use of a column with a particle size less than 2 μmin liquid chromatography. In some embodiments, UPLC refers to the use ofa high linear solvent velocity (e.g., as observed when operating at 6000psi or higher) in liquid chromatography. Exemplary UPLC machines arecommercially available (e.g., the ACQUITY UPLC® from Waters; Milford,Mass.).

In some embodiments, mass spectrometry (e.g., used in LC/MS as describedherein) may refer to electrospray ionization mass spectrometry (ESI-MS).ESI-MS is known in the art as a technique that uses electrical energy toanalyze ions derived from a solution using mass spectrometry (see, e.g.,Yamashita, M. and Fenn, J. B. (1984) J. Phys. Chem. 88:4451-4459). Ionicspecies (or neutral species that are ionized in solution or in gaseousphase) are transferred from a solution to a gaseous phase by dispersalin an aerosol of charged droplets, followed by solvent evaporation thatreduces the size of the charged droplets and sample ion ejection fromthe charge droplets as the solution is passed through a small capillarywith a voltage relative to ground (e.g., the wall of the surroundingchamber). In some embodiments, the capillary voltage is from about 2 kVto about 10 kV, or about 2.5 kV to about 6.0 kV. In certain embodiments,liquid chromatography (e.g., used in LC/MS as described herein) uses acapillary voltage of about 3.5 kV. In some embodiments the capillaryvoltage ranges from about 1 kV to about 10 kV. In other embodiments,mass spectrometry (e.g., used in LC/MS as described herein) may refer tomatrix-assisted laser desorption/ionization (MALDI).

In some embodiments, mass spectrometry (e.g., used in LC/MS as describedherein) uses a sampling cone and/or skimmer, through which ions may passbefore entering the analyzer. In some embodiments, e.g., when applyingvoltage to the capillary as described above, the sample cone is held ata lower voltage than the capillary voltage. In certain embodiments,liquid chromatography (e.g., used in LC/MS as described herein) uses asampling cone voltage of about 45 V. In some embodiments the samplingcone voltage ranges from about 0 V to about 200 V.

In some embodiments, mass spectrometry (e.g., used in LC/MS as describedherein) uses assisted calibration. Calibration, when used in referenceto mass spectrometry, may include the introduction of one or morecompounds having a known mass (e.g., a standard) for the purpose ofcalibrating the instrument with respect to mass detection (e.g., m/zmeasurements). In some embodiments, assisted calibration may refer tousing software to correlate a peak and/or position of a known standard(e.g., a calibrant) to a specific mass-to-charge (m/z) ratio. Oncecalibrated, the user may then perform mass spectrometry on a samplehaving one or more unknown compounds, or compounds present at an unknownconcentration, within a certain degree of accuracy or error, and/or adesired level of reproducibility, e.g., as compared to a previous orknown experimental condition. Various calibrants are known in the art,including without limitation sodium iodide, sodium cesium iodide,polyethylene glycol, and perfluorotributylamine. In certain embodiments,sodium iodide is used as a calibrant. In some embodiments the calibrantsare Glu-1-fibrinopeptide B and leucine encephalin peptide to lock massduring LC/MS operation.

In some embodiments, the methods include subjecting a denatured viralparticle of the present disclosure, or subjecting digested fragments ofa denatured viral particle of the present disclosure, to liquidchromatography/mass spectrometry-mass spectrometry (LC/MS/MS). As isknown in the art, LC/MS/MS (the term “liquid chromatography-tandem massspectrometry” may be used interchangeably herein) utilizes liquidchromatography for physical separation of ions and mass spectrometry forgeneration of mass spectral data from the ions, where the massspectrometry uses multiple stages of mass (e.g., m/z) separation,typically separated by a fragmentation step. For example, ions ofinterest within a range of m/z may be separated out in a first round ofMS, fragmented, and then further separated based on individual m/z in asecond round of MS. Ion fragmentation may include without limitation atechnique such as collision-induced dissociation (CID), higher energycollision dissociation (HCD), electron-capture dissociation (ECD), orelectron-transfer dissociation (ETD).

In some embodiments, the methods include subjecting a denatured viralparticle of the present disclosure to reduction and/or alkylation. Meansto reduce the viral particle include but are not limited to treatmentwith dithiothreitol, β-mercaptoethanol, or tris(2-carboxyethyl)phosphine(TCEP). Means to alkylate the viral particle include but are not limitedto treating the AAV particle with iodoacetic acid, iodoacetamide, or4-vinylpyridine.

In some embodiments, the methods include subjecting a denatured viralparticle of the present disclosure to digestion, e.g., to generatefragments of VP1, VP2 and/or VP3 of an AAV particle. For example, adenatured AAV particle may be subjected to digestion to generate peptidefragments that may be analyzed, e.g., using LC for separation and MS/MSfor analysis (see below for greater description). In some embodiments,the digestion is an enzymatic digestion. In some embodiments, thedigestion uses chemical digestion such as CNBr treatment of instrumentfragmentation (e.g., top down). In some embodiments, the digestion useschemical digestion such as acid digestion.

In some embodiments, the enzymatic digestion is an endopeptidasedigestion. An endopeptidase may include any peptidase that catalyzes theproteolysis of peptide bonds of non-terminal amino acids of apolypeptide. Known endopeptidases may include, without limitation,trypsin, chymotrypsin, AspN, Glu-C, LysC, pepsin, thermolysin, glutamylendopeptidase, elastase, and neprilysin. In certain embodiments, theenzymatic digestion is a trypsin digestion or a LysC digestion.

In some embodiments, the liquid chromatography (e.g., used in LC/MS orLC/MS/MS as described herein) is reverse phase liquid chromatography(the terms “reversed phase liquid chromatography” or RPLC may be usedinterchangeably herein with reverse phase liquid chromatography). As isknown in the art, reverse phase liquid chromatography may refer to achromatographic separation using a hydrophobic stationary phase (e.g., asupport or substrate such as a column) to adsorb hydrophobic moleculesin a polar mobile phase. By decreasing the polarity of the mobile phase(e.g., by adding an organic solvent), one may achieve gradientseparation of molecules by hydrophobicity, since more hydrophobicmolecules will stay on the column in higher concentrations of organicsolvent due to stronger hydrophobic interactions with the column. Insome embodiment, separation is by capillary electrophoresis (CE), sizeexclusion chromatography (SEC), ion exchange chromatography (IEC) suchas cation exchange chromatography, hydrophobic interactionchromatography (HIC), hydrophilic interaction liquid chromatography(HILIC), but not limited to on-line LC/MS such as offline separationbefore MS; e.g., tips, columns; plates or cartridges.

Generally, a stationary phase suitable for reverse phase liquidchromatography (e.g., a hydrophobic moiety) may be coupled to a supportincluding without limitation a column or resin packed with particles orbeads (e.g., porous silica particles or polystyrene). A variety ofhydrophobic stationary phases are known in the art, including withoutlimitation hydrophobic alkyl chains, octyl or octadecyl silyl moieties,cyano moieties, and amino moieties. In some embodiments, the stationaryphase may include a hydrophobic alkyl chain of a particular length, suchas C4, C8, or C18. In certain embodiments, the reverse phasechromatography is a C4 or C8 reverse chromatography (e.g., reverse phasechromatography utilizing a C4 or C8 stationary phase). One of skill inthe art may suitably select a stationary phase based on the molecule ofinterest (e.g., a denatured AAV particle or fragment thereof).

A variety of mobile phases suitable for reverse phase liquidchromatography are known in the art. As described above, a reverse phaseliquid chromatography mobile phase may include a mixture of organic(e.g., hydrophobic) and aqueous (e.g., polar) solvents. Increasing theproportion of organic solvent increases its power to elute hydrophobiccompounds from the stationary phase. Compound retention and/orselectivity may be altered, e.g., by changing the type or exposure ofthe stationary phase, adding polar reagents such as end cappingreagents, altering the temperature, and/or altering mobile phasecharacteristics such as the proportion of organic solvent, pH, buffers,and the type of organic solvent used. In some embodiments, the polarcomponent of the mobile phase may include without limitation water or anaqueous buffer. In some embodiments, the polar component of the mobilephase may include without limitation acetonitrile, methanol, ethanol,isopropyl alcohol, tetrahydrofuran (THF), and formic acid.

In some embodiments, two or more mobile phases may be used (e.g., mobilephase A, mobile phase B, etc.) in a gradient or proportion of interest.In certain embodiments, the chromatography uses a mobile phase Acomprising formic acid in water. In certain embodiments, the mobilephase A comprises about 0.1% formic acid. In certain embodiments, themobile phase A comprises about 0.1% to about 5% formic acid. In certainembodiments, the chromatography uses a mobile phase B comprising formicacid in acetonitrile. In certain embodiments, the mobile phase Bcomprises about 0.1% formic acid.

In some embodiments, the proportion of mobile phase B in thechromatography increases over time. For example, the proportion ofmobile phase B in the chromatography may be increased in a stepwisemanner. In certain embodiments, mobile phase B increases from about 10%to about 20%, from about 20% to about 30%, and from about 30% to about38%. In other embodiments, mobile phase B increases from about 2% toabout 60%. In other embodiments, mobile phase B increases from about 2%to about 100% from about 1 min to about 200 min In some embodiments, theremainder of the mobile phase is a second mobile phase of the presentdisclosure, e.g., mobile phase A. In certain embodiments, mobile phase Bincreases from about 10% to about 20% in about 6 minutes, from about 20%to about 30% in about 10 minutes, and from about 30% to about 38% inabout 40 minutes. In other embodiments, mobile phase B increases fromabout 2% to about 60% in about 121 minutes. One of skill in the art maysuitably adjust the mobile phase of interest and the gradient timingused based on the desired chromatographic separation and/or analyte ofinterest.

In some embodiments, the liquid chromatography is high-performanceliquid chromatography (HPLC). HPLC is known in the art as a form ofliquid chromatography in which a liquid solvent containing a sample ispressurized as it passes through a column containing solid phase. Whiletraditional or low pressure LC may use gravity to pass a mobile phasethrough the solid phase, HPLC uses pumps to apply a pressure to themobile phase and typically uses a solid phase with smaller particles toincrease resolution. In some embodiments, the HPLC uses a pressure ofbetween about 50 bar and about 350 bar. In some embodiments, reversedphase HPLC may be used to concentrate and/or desalt proteins (e.g., AAVcapsid proteins) for MS analysis.

In some embodiments, one or more parameters including without limitationsource voltage, capillary temperature, ESI voltage (if using ESI-MS),CID energy, and the number of MS/MS events may be adjusted, e.g., inLC/MS/MS as used herein, based on the findings described herein. In someembodiments, mass spectrometry (e.g., used in LC/MS/MS as describedherein) uses a source voltage (e.g., capillary voltage) of about 2.5 kV.In some embodiments, mass spectrometry (e.g., used in LC/MS/MS asdescribed herein) uses a capillary temperature of about 275° C. In someembodiments, the capillary temperature ranges from about 20° C. to about400° C.

A variety of mass analyzers suitable for LC/MS and/or LC/MS/MS are knownin the art, including without limitation time-of-flight (TOF) analyzers,quadrupole mass filters, quadrupole TOF (QTOF), and ion traps (e.g., aFourier transform-based mass spectrometer or an Orbitrap). In Orbitrap,a barrel-like outer electrode at ground potential and a spindle-likecentral electrode are used to trap ions in trajectories rotatingelliptically around the central electrode with oscillations along thecentral axis, confined by the balance of centrifugal and electrostaticforces. The use of such instruments employs a Fourier transformoperation to convert a time domain signal (e.g., frequency) fromdetection of image current into a high resolution mass measurement(e.g., nano LC/MS/MS). Further descriptions and details may be found,e.g., in Scheltema, R. A. et al. (2014) Mol. Cell Proteomics13:3698-3708; Perry, R. H. et al. (2008) Mass. Spectrom. Rev.27:661-699; and Scigelova, M. et al. (2011) Mol. Cell Proteomics10:M111.009431.

As described above, in some embodiments, the MS includes nano LC/MS/MS,e.g., using an Orbitrap mass analyzer. In some embodiments, the ionsource may include a stacked-ring ion guide or S-lens. As is known inthe art, an S-lens may be employed to focus the ion beam using radiofrequency (RF), thereby increasing transmission of ions into theinstrument. This may improve sensitivity (e.g., for low-intensity ions)and/or improve the scan rate. In certain embodiments, the S-lens RFlevel of the mass spectrometry is about 55%. In certain embodiments, theS-lens RF level of the mass spectrometry is about 20% to about 100%.

In some embodiments, masses of viral capsid proteins may be determined,e.g., based on LC/MS and/or LC/MS/MS data. In some embodiments, massesof VP1, VP2 and VP3 of an AAV particle, or of fragments of VP1, VP2 andVP3 of the AAV particle, may be determined, e.g., based on LC/MS and/orLC/MS/MS data. Various methods to determine protein mass and/or identityfrom MS data are known in the art. For example, peptide massfingerprinting may be used to determine protein sequence based on MSdata, or proteins may be identified based on MS/MS data related to oneor more constituent peptides. When using tandem MS, product ion scanningmay be used to analyze m/z data related to one or more peptides of aprotein of interest. Software known in the art may then be used, e.g.,to match identified peaks to reference or known peaks, to group peaksinto isotopomer envelopes, and so forth. Peptide mass values may becompared to a database of known peptide sequences. For example, Mascotmay be used to match observed peptides with theoretical databasepeptides, e.g., resulting from application of a particular digestpattern to an in silico protein database. Other suitable software mayinclude without limitation Proteome Discoverer, ProteinProspector,X!Tandem, Pepfinder, Bonics, or MassLynx™ (Waters). Other softwaresuitable for various steps of MS data analysis may be found, e.g., atwww.ms-utils.org/wiki/pmwiki.php/Main/SoftwareList.

In some embodiments, a determined or calculated mass of the presentdisclosure (e.g., the determined or calculated mass of VP1, VP2 and/orVP3 of the AAV particle) may be compared with a reference, e.g., atheoretical mass of a VP1, VP2, and/or VP3 of one or more AAV serotypes.A reference of the present disclosure may include a theoretical mass ofa VP1, VP2, and/or VP3 of one or more of any of the AAV serotypesdescribed herein. For example, in some embodiments, the masses of VP1,VP2, and/or VP3 are compared to the theoretical masses of one or more ofan AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5capsid, an AAV6 capsid, an AAV7 capsid, an AAV8 capsid, an AAVrh8capsid, an AAV9 capsid, an AAV10 capsid, an AAVrh10 capsid, an AAV11capsid, an AAV12 capsid, an AAV LK03 capsid (see U.S. Pat. No.9,169,299), an AAV2R471A capsid, an AAV2/2-7m8 capsid, an AAV DJ capsid(see U.S. Pat. No. 7,588,772), an AAV DJ8 capsid, an AAV2 N587A capsid,an AAV2 E548A capsid, an AAV2 N708A capsid, an AAV V708K capsid, a goatAAV capsid, an AAV1/AAV2 chimeric capsid, a bovine AAV capsid, or amouse AAV capsid rAAV2/HBoV1 (chimeric AAV/human bocavirus virus 1), anAAV2HBKO capsid, an AAVPHP.B capsid or an AAVPHP.eB capsid. In someembodiments, a determined or calculated mass of the present disclosure(e.g., the determined or calculated mass of VP1, VP2 and/or VP3 of theAAV particle) may be compared with a theoretical mass of a VP1, VP2,and/or VP3 of the corresponding AAV serotype.

In some embodiments, the methods of the present disclosure may includedetermining the heterogeneity of an AAV particle. In some embodiments, adeviation of one or more of the masses of VP1, VP2 and/or VP3 (e.g.,from a reference mass, such as a theoretical, predicted, or expectedmass) is indicative of the AAV capsid heterogeneity. In someembodiments, heterogeneity may include one or more of the following,without limitation: mixed serotypes, variant capsids, capsid amino acidsubstitutions, truncated capsids, or modified capsids.

In some embodiments, the use of LC/MS and LC/MS/MS as described hereinmay be combined. In some embodiments, a method of determining theserotype of an AAV particle may include subjecting a denatured AAVparticle to LC/MS (e.g., as described herein) and determining the massesof VP1, VP2 and VP3 of the AAV particle; as well as subjecting fragmentsof VP1, VP2 and/or VP3 to LC/MS/MS and determining the masses offragments of VP1, VP2 and VP3 of the AAV particle (the specificcombination of masses of fragments of VP1, VP2 and VP3 are indicative ofthe AAV serotype). In some embodiments, a method of determining theheterogeneity of an AAV particle may include subjecting a denatured AAVparticle to LC/MS (e.g., as described herein), determining the masses ofVP1, VP2 and VP3 of the AAV particle, and comparing these masses withthe theoretical masses of VP1, VP2 and VP3 of the AAV serotype; as wellas subjecting fragments of VP1, VP2 and/or VP3 to LC/MS/MS, determiningthe masses of fragments of VP1, VP2 and VP3 of the AAV particle, andcomparing these masses with the theoretical masses of VP1, VP2 and VP3of the AAV serotype (a deviation of one or more of the masses of VP1,VP2 or VP3 are indicative of the AAV capsid heterogeneity).

In some embodiments, an AAV particle of the present disclosure may beacetylated. For example, in some embodiments, the N-terminus of VP1and/or VP3 is acetylated. As described in greater detail below, theamino acid at the 2^(nd) position to the initiating methionine (iMet X)of an AAV capsid protein may be mutated in order to determine its effecton N-terminal (Nt-) acetylation, as well as the functional consequencesof affecting Nt-acetylation on AAV particle trafficking, transduction,and/or post-translational modification (e.g., glycosylation,ubiquitination, and so forth). In some embodiments, the N-terminus of anAAV capsid protein (e.g., VP1 or VP3) may refer to the first amino acidafter the initiating methionine, which in some cases may be removed by,e.g., a Met-aminopeptidase.

In some embodiments, an AAV particle of the present disclosure (e.g., arecombinant AAV or rAAV particle) comprises an amino acid substitutionat amino acid residue 2 of VP1 and/or VP3. In some embodiments, theamino acid substitution at amino acid residue 2 of VP1 and/or VP3 leadsto a VP1 and/or VP3 with a different frequency or proportion ofN-terminal acetylation as compared to a reference (e.g., the parent AAVparticle before the amino acid substitution, or an AAV particle with adifferent amino acid residue 2 of VP1 and/or VP3). In some embodiments,the amino acid substitution at amino acid residue 2 of VP1 and/or VP3alters N-terminal acetylation as compared to N-terminal acetylation atamino acid residue 2 of VP1 and/or VP3 of the parent AAV particle. Forexample, in certain embodiments, the amino acid substitution at aminoacid residue 2 of VP1 alters N-terminal acetylation as compared toN-terminal acetylation at amino acid residue 2 of VP1 of the parent AAVparticle. In certain embodiments, the amino acid substitution at aminoacid residue 2 of VP3 alters N-terminal acetylation as compared toN-terminal acetylation at amino acid residue 2 of VP3 of the parent AAVparticle. In some embodiments, an amino acid substitution (e.g., anamino acid substitution at amino acid residue 2 of VP1 or VP3) that“alters” N-terminal acetylation results in a higher frequency ofN-terminal acetylation or a lower frequency of N-terminal acetylation,e.g., as compared to a VP1 or VP3 without the substitution, such as theparental VP1 or VP3. The VP1 and/or VP3 may belong to any of theexemplary AAV serotypes described herein, including variants or hybridsthereof (e.g., bearing tyrosine mutation or heparin binding mutations).Exemplary assays for N-terminal acetylation include without limitationmass spectrometry, isotope labeling (e.g., with an isotope-labeledacetyl group or precursor thereof), Western blotting with anacetylation-specific antibody, and so forth. In some embodiments, aminoacid residue 2 of the AAV capsid protein (e.g., VP1 or VP3) issubstituted with Cys, Ser, Thr, Val, Gly, Asn, Asp, Glu, Ile, Leu, Phe,Gln, Lys, Met, Pro or Tyr. In some embodiments, the amino acidsubstitution results in less deamidation of the AAV capsid.

In some embodiments, an AAV particle of the present disclosure may bedeamidated. For example, in some embodiments, N57 of VP1 and/or N382,N511, and/or N715 VP3 is deamidated. As described in greater detailbelow, an amino acid selected from A35 of VP1, N57 of VP1, G58 of VP1,N382 of VP3, G383 of VP3, N511 of VP3, G512 of VP3, N715 of VP3, or G716of VP3 of an AAV capsid protein (e.g., VP1 or VP3) may be mutated inorder to determine its effect on deamidation, as well as the functionalconsequences of affecting deamidation on AAV particle trafficking,transduction, and/or post-translational modification (e.g.,glycosylation, ubiquitination, and so forth).

In some embodiments, an AAV particle of the present disclosure (e.g., arecombinant AAV or rAAV particle) comprises an amino acid substitutionat one or more amino acid residues selected from A35 of VP1, N57 of VP1,G58 of VP1, N382 of VP3, G383 of VP3, N511 of VP3, G512 of VP3, N715 ofVP3, and G716 of VP3. In some embodiments, the amino acid substitutionat A35 of VP1, N57 of VP1, G58 of VP1, N382 of VP3, G383 of VP3, N511 ofVP3, G512 of VP3, N715 of VP3, and/or G716 of VP3 leads to a VP1 and/orVP3 with a different frequency or proportion of deamidation as comparedto a reference (e.g., the parent AAV particle before the amino acidsubstitution, or an AAV particle with a different corresponding aminoacid residue 2). In some embodiments, an amino acid substitution (e.g.,an amino acid substitution at A35 of VP1, N57 of VP1, G58 of VP1, N382of VP3, G383 of VP3, N511 of VP3, G512 of VP3, N715 of VP3, or G716 ofVP3) that “alters” deamidation results in a higher frequency ofdeamidation or a lower frequency of deamidation, e.g., as compared to aVP1 or VP3 without the substitution, such as the parental VP1 or VP3.The VP1 and/or VP3 may belong to any of the exemplary AAV serotypesdescribed herein, including variants or hybrids thereof (e.g., bearingtyrosine mutation or heparin binding mutations). Exemplary assays fordeamidation include without limitation mass spectrometry, HPLC (see,e.g., the ISOQUANT® isoaspartate detection kit from Promega), and soforth. In some embodiments, N57 of VP1, N382 of VP3, N511 of VP3, and/orN715 of VP3 is substituted with Asp, and the amino acid substitutionresults in a higher frequency of deamidation as compared to deamidationof VP1 and/or VP3 of the parent AAV particle. In other embodiments, theamino acid substitution is N57K or N57Q, and the amino acid substitutionresults in a lower frequency of deamidation as compared to deamidationof VP1 and/or VP3 of the parent AAV particle. In yet other embodiments,G58 of VP1, G383 of VP3, G512 of VP3, and/or G716 of VP3 is substitutedwith an amino acid that is not Gly (e.g., Ala, Arg, Asn, Asp, Cys, Glu,Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val), andthe amino acid substitution results in a lower frequency of deamidationas compared to deamidation of VP1 and/or VP3 of the parent AAV particle.In yet other embodiments, A35 of VP1 is substituted with Asn and resultsin a higher frequency of deamidation as compared to deamidation of VP1of a parent particle.

As used herein “N-acetylation” refers to a process whereby an acetylgroup is covalently added to the amino group of the N-terminal aminoacid of a protein. Typically, N-terminal acetyltransferases (NATs)transfer an acetyl group from acetyl-coenzyme A (Ac-CoA) to the α-aminogroup of the first amino acid residue of the protein.

As used here in, “deamidation” refers to a chemical reaction in which anamide functional group in the side chain of asparagine or glutamine isremoved or converted to another functional group. For example,asparagine may be converted to aspartic acid or isoaspartic acid. Inother examples, glutamine is converted to glutamic acid or pyroglutamicacid (5-oxoproline).

In some embodiments, the AAV particle is N-acetylated to a higher extentcompared to a parental AAV capsid protein. In some embodiments, the AAVparticle comprises more than about any of 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%more N-acetyl groups compared to a parent AAV particle. In someembodiments, the AAV particle comprises between about any of 5%-10%,10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-55%, 45%-50%,50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%,90%-95%, 95%-100%, 5-25%, 25-50%, 50-75%, 75%-100%, 5-50% or 50%-100%more N-acetyl groups compared to a parent AAV particle. In someembodiments, the AAV particle comprises more than about any of 2-fold,3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 500-fold,or 1000-fold more N-acetyl groups compared to a parent AAV particle. Insome embodiments, the AAV particle comprises between about any of 2-foldto 3-fold, 3-fold to 4-fold, 4-fold to 5-fold, 5-fold to 10-fold,10-fold to 25-fold, 25-fold to 50-fold, 50-fold to 100-fold, 100-fold to500-fold, 500-fold to 1000-fold, 2-fold to 10-fold, 10-fold to 100-fold,or 100-fold to 1000-fold more N-acetyl groups compared to a parent AAVparticle.

In some embodiments, the AAV particle N-acetlyated to a lower extentcompared to a parental AAV capsid protein. In some embodiments, the AAVparticle comprises more than about any of 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%less N-acetyl groups compared to a parent AAV particle. In someembodiments, the AAV particle comprises between about any of 5%-10%,10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-55%, 45%-50%,50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%,90%-95%, 95%-100%, 5-25%, 25-50%, 50-75%, 75%-100%, 5-50% or 50%-100%less N-acetyl groups compared to a parent AAV particle. In someembodiments, the AAV particle comprises more than about any of 2-fold,3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 500-fold,or 1000-fold less N-acetyl groups compared to a parent AAV particle. Insome embodiments, the AAV particle comprises between about any of 2-foldto 3-fold, 3-fold to 4-fold, 4-fold to 5-fold, 5-fold to 10-fold,10-fold to 25-fold, 25-fold to 50-fold, 50-fold to 100-fold, 100-fold to500-fold, 500-fold to 1000-fold, 2-fold to 10-fold, 10-fold to 100-fold,or 100-fold to 1000-fold less N-acetyl groups compared to a parent AAVparticle.

In some embodiments, the AAV particle is deamidated to a higher extentcompared to a parental AAV particle. In some embodiments, the AAVparticle is more than about any of 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% moredeamidated compared to a parent AAV particle. In some embodiments, theAAV particle is deamidated between about any of 5%-10%, 10%-15%,15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-55%, 45%-50%, 50%-55%,55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%,95%-100%, 5-25%, 25-50%, 50-75%, 75%-100%, 5-50% or 50%-100% more than aparent AAV particle. In some embodiments, the AAV particle is deamidatedmore than about any of 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold,50-fold, 100-fold, 500-fold, or 1000-fold compared to a parent AAVparticle. In some embodiments, the AAV particle is deamidated betweenabout any of 2-fold to 3-fold, 3-fold to 4-fold, 4-fold to 5-fold,5-fold to 10-fold, 10-fold to 25-fold, 25-fold to 50-fold, 50-fold to100-fold, 100-fold to 500-fold, 500-fold to 1000-fold, 2-fold to10-fold, 10-fold to 100-fold, or 100-fold to 1000-fold more than aparent AAV particle.

In some embodiments, a capsid protein of AAV is deamidated to a lowerextent compared to a parental AAV capsid protein. In some embodiments,the AAV particle is more than about any of 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%less deamidated compared to a parent AAV particle. In some embodiments,the AAV particle is deamidated between about any of 5%-10%, 10%-15%,15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-55%, 45%-50%, 50%-55%,55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%,95%-100%, 5-25%, 25-50%, 50-75%, 75%-100%, 5-50% or 50%-100% less than aparent AAV particle. In some embodiments, the AAV particle is deamidatedmore than about any of 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold,50-fold, 100-fold, 500-fold, or 1000-fold less than a parent AAVparticle. In some embodiments, the AAV particle is deamidated betweenabout any of 2-fold to 3-fold, 3-fold to 4-fold, 4-fold to 5-fold,5-fold to 10-fold, 10-fold to 25-fold, 25-fold to 50-fold, 50-fold to100-fold, 100-fold to 500-fold, 500-fold to 1000-fold, 2-fold to10-fold, 10-fold to 100-fold, or 100-fold to 1000-fold less than aparent AAV particle.

The invention provides any combination of N-acetylation and deamidation.For example, the AAV capsid protein may be N-acetylated to a higherextent than a parent AAV capsid protein and deamidated to a higherextent than a parent AAV capsid protein, the AAV capsid protein may beN-acetylated to a higher extent than a parent AAV capsid protein anddeamidated to the same extent than a parent AAV capsid protein, the AAVcapsid protein may be N-acetylated to a higher extent than a parent AAVcapsid protein and deamidated to a lower extent than a parent AAV capsidprotein, the AAV capsid protein may be N-acetylated to the same extentthan a parent AAV capsid protein and deamidated to a higher extent thana parent AAV capsid protein, the AAV capsid protein may be N-acetylatedto the same extent than a parent AAV capsid protein and deamidated tothe same extent than a parent AAV capsid protein, the AAV capsid proteinmay be N-acetylated to the same extent than a parent AAV capsid proteinand deamidated to a lower extent than a parent AAV capsid protein, theAAV capsid protein may be N-acetylated to a lower extent than a parentAAV capsid protein and deamidated to a higher extent than a parent AAVcapsid protein, the AAV capsid protein may be N-acetylated to a lowerextent than a parent AAV capsid protein and deamidated to the sameextent than a parent AAV capsid protein, or the AAV capsid protein maybe N-acetylated to a lower extent than a parent AAV capsid protein anddeamidated to a lower extent than a parent AAV capsid protein.

IV. Vectors

In certain aspects, the invention relates to viral particles, suitablefor use in any of the methods described herein, which may comprise AAVvectors (e.g., rAAV vectors) or vectors derived from another virus. Insome embodiments, the viral particle comprises a vector encoding aheterologous nucleic acid, e.g., a heterologous transgene. In someembodiments, the AAV particle comprises an AAV vector genome encoding aheterologous nucleic acid, e.g., a heterologous transgene.

The present invention contemplates the use of a recombinant viral genomefor introduction of one or more nucleic acid sequences encoding atherapeutic polypeptide and/or nucleic acid for packaging into a rAAVviral particle. The recombinant viral genome may include any element toestablish the expression of the therapeutic polypeptide and/or nucleicacid, for example, a promoter, an ITR of the present disclosure, aribosome binding element, terminator, enhancer, selection marker,intron, polyA signal, and/or origin of replication.

In some embodiments, the heterologous nucleic acid encodes a therapeuticpolypeptide. A therapeutic polypeptide may, e.g., supply a polypeptideand/or enzymatic activity that is absent or present at a reduced levelin a cell or organism. Alternatively, a therapeutic polypeptide maysupply a polypeptide and/or enzymatic activity that indirectlycounteracts an imbalance in a cell or organism. For example, atherapeutic polypeptide for a disorder related to buildup of ametabolite caused by a deficiency in a metabolic enzyme or activity maysupply a missing metabolic enzyme or activity, or it may supply analternate metabolic enzyme or activity that leads to reduction of themetabolite. A therapeutic polypeptide may also be used to reduce theactivity of a polypeptide (e.g., one that is overexpressed, activated bya gain-of-function mutation, or whose activity is otherwisemisregulated) by acting, e.g., as a dominant-negative polypeptide.

The nucleic acids of the invention may encode polypeptides that areintracellular proteins, anchored in the cell membrane, remain within thecell, or are secreted by the cell transduced with the vectors of theinvention. For polypeptides secreted by the cell that receives thevector; the polypeptide can be soluble (i.e., not attached to the cell).For example, soluble polypeptides are devoid of a transmembrane regionand are secreted from the cell. Techniques to identify and removenucleic acid sequences which encode transmembrane domains are known inthe art.

The nucleic acids if the invention (e.g. the AAV vector genome) maycomprise as a transgene, a nucleic acid encoding a protein or functionalRNA that modulates or treats a CNS-associated disorder. The following isa non-limiting list of genes associated with CNS-associated disorders:neuronal apoptosis inhibitory protein (NAIP), nerve growth factor (NGF),glial-derived growth factor (GDNF), brain-derived growth factor (BDNF),ciliary neurotrophic factor (CNTF), tyrosine hydroxylase (TM,GTP-cyclohydrolase (GTPCH), aspartoacylase (ASPA), superoxide dismutase(SOD1), an anti-oxidant, an anti-angiogenic polypeptide, ananti-inflammatory polypeptide, and amino acid decorboxylase (AADC). Forexample, a useful transgene in the treatment of Parkinson's diseaseencodes TH, which is a rate limiting enzyme in the synthesis ofdopamine. A transgene encoding GTPCII, which generates the TII cofactortetrahydrobiopterin, may also be used in the treatment of Parkinson'sdisease. A transgene encoding GDNF or BDNF, or AADC, which facilitatesconversion of L-Dopa to DA, may also be used for the treatment ofParkinson's disease. For the treatment of ALS, a useful transgene mayencode: GDNF, BDNF or CNTF. Also for the treatment of ALS, a usefultransgene may encode a functional RNA, e.g., shRNA, miRNA, that inhibitsthe expression of SOD1. For the treatment of ischemia a useful transgenemay encode NAIP or NGF. A transgene encoding Beta-glucuronidase (GUS)may be useful for the treatment of certain lysosomal storage diseases(e.g., Mucopolysacharidosis type VII (MPS VII)). A transgene encoding aprodrug activation gene, e.g., HSV-Thymidine kinase which convertsganciclovir to a toxic nucleotide which disrupts DNA synthesis and leadsto cell death, may be useful for treating certain cancers, e.g., whenadministered in combination with the prodrug. A transgene encoding anendogenous opioid, such a β-endorphin may be useful for treating pain.Examples of anti-oxidants include without limitation SOD1; SOD2;Catalase; Sirtuins 1, 3, 4, or 5; NRF2; PGC1a; GCL (catalytic subunit);GCL (modifier subunit); adiponectin; glutathione peroxidase 1; andneuroglobin. Examples of anti-angiogenic polypeptides include withoutlimitation angiostatin, endostatin, PEDF, a soluble VEGF receptor, and asoluble PDGF receptor. Examples of anti-inflammatory polypeptidesinclude without limitation IL-10, soluble IL17R, soluble TNF-R,TNF-R-Ig, an IL-1 inhibitor, and an IL18 inhibitor. Other examples oftransgenes that may be used in the rAAV vectors of the invention will beapparent to the skilled artisan (See, e.g., Costantini L C, et al., GeneTherapy (2000) 7, 93-109).

In some embodiments, the heterologous nucleic acid encodes a therapeuticnucleic acid. In some embodiments, a therapeutic nucleic acid mayinclude without limitation an siRNA, an shRNA, an RNAi, a miRNA, anantisense RNA, a ribozyme or a DNAzyme. As such, a therapeutic nucleicacid may encode an RNA that when transcribed from the nucleic acids ofthe vector can treat a disorder by interfering with translation ortranscription of an abnormal or excess protein associated with adisorder of the invention. For example, the nucleic acids of theinvention may encode for an RNA which treats a disorder by highlyspecific elimination or reduction of mRNA encoding the abnormal and/orexcess proteins. Therapeutic RNA sequences include RNAi, smallinhibitory RNA (siRNA), micro RNA (miRNA), and/or ribozymes (such ashammerhead and hairpin ribozymes) that can treat disorders by highlyspecific elimination or reduction of mRNA encoding the abnormal and/orexcess proteins.

In some embodiments, the therapeutic polypeptide or therapeutic nucleicacid is used to treat a disorder of the CNS. Without wishing to be boundto theory, it is thought that a therapeutic polypeptide or therapeuticnucleic acid may be used to reduce or eliminate the expression and/oractivity of a polypeptide whose gain-of-function has been associatedwith a disorder, or to enhance the expression and/or activity of apolypeptide to complement a deficiency that has been associated with adisorder (e.g., a mutation in a gene whose expression shows similar orrelated activity). Non-limiting examples of disorders of the inventionthat may be treated by a therapeutic polypeptide or therapeutic nucleicacid of the invention (exemplary genes that may be targeted or suppliedare provided in parenthesis for each disorder) include stroke (e.g.,caspase-3, Beclin1, Ask1, PAR1, HIF1α, PUMA, and/or any of the genesdescribed in Fukuda, A. M. and Badaut, J. (2013) Genes (Basel)4:435-456), Huntington's disease (mutant HTT), epilepsy (e.g., SCN1A,NMDAR, ADK, and/or any of the genes described in Boison, D. (2010)Epilepsia 51:1659-1668), Parkinson's disease (alpha-synuclein), LouGehrig's disease (also known as amyotrophic lateral sclerosis; SOD1),Alzheimer's disease (tau, amyloid precursor protein), corticobasaldegeneration or CBD (tau), corticogasal ganglionic degeneration or CBGD(tau), frontotemporal dementia or FTD (tau), progressive supranuclearpalsy or PSP (tau), multiple system atrophy or MSA (alpha-synuclein),cancer of the brain (e.g., a mutant or overexpressed oncogene implicatedin brain cancer), and lysosomal storage diseases (LSD). Disorders of theinvention may include those that involve large areas of the cortex,e.g., more than one functional area of the cortex, more than one lobe ofthe cortex, and/or the entire cortex. Other non-limiting examples ofdisorders of the invention that may be treated by a therapeuticpolypeptide or therapeutic nucleic acid of the invention includetraumatic brain injury, enzymatic dysfunction disorders, psychiatricdisorders (including post-traumatic stress syndrome), neurodegenerativediseases, and cognitive disorders (including dementias, autism, anddepression). Enzymatic dysfunction disorders include without limitationleukodystrophies (including Canavan's disease) and any of the lysosomalstorage diseases described below.

In some embodiments, the therapeutic polypeptide or therapeutic nucleicacid is used to treat a lysosomal storage disease. As is commonly knownin the art, lysosomal storage disease are rare, inherited metabolicdisorders characterized by defects in lysosomal function. Such disordersare often caused by a deficiency in an enzyme required for propermucopolysaccharide, glycoprotein, and/or lipid metabolism, leading to apathological accumulation of lysosomally stored cellular materials.Non-limiting examples of lysosomal storage diseases of the inventionthat may be treated by a therapeutic polypeptide or therapeutic nucleicacid of the invention (exemplary genes that may be targeted or suppliedare provided in parenthesis for each disorder) include Gaucher diseasetype 2 or type 3 (acid beta-glucosidase, GBA), GM1 gangliosidosis(beta-galactosidase-1, GLB1), Hunter disease (iduronate 2-sulfatase,IDS), Krabbe disease (galactosylceramidase, GALC), a mannosidosisdisease (a mannosidase, such as alpha-D-mannosidase, MAN2B1), βmannosidosis disease (beta-mannosidase, MANBA), metachromaticleukodystrophy disease (pseudoarylsulfatase A, ARSA),mucolipidosisII/III disease (N-acetylglucosamine-1-phosphotransferase,GNP TAB), Niemann-Pick A disease (acid sphingomyelinase, ASM),Niemann-Pick C disease (Niemann-Pick C protein, NPC1), Pompe disease(acid alpha-1,4-glucosidase, GAA), Sandhoff disease (hexosaminidase betasubunit, HEXB), Sanfilippo A disease (N-sulfoglucosamine sulfohydrolase,MPS3A), Sanfilippo B disease (N-alpha-acetylglucosaminidase, NAGLU),Sanfilippo C disease (heparin acetyl-CoA:alpha-glucosaminidaseN-acetyltransferase, MP S3C), Sanfilippo D disease(N-acetylglucosamine-6-sulfatase, GNS), Schindler disease(alpha-N-acetylgalactosaminidase, NAGA), Sly disease(beta-glucuronidase, GUSB), Tay-Sachs disease (hexosaminidase alphasubunit, HEXA), and Wolman disease (lysosomal acid lipase, LIPA).

Additional lysosomal storage diseases, as well as the defective enzymeassociated with each disease, are listed in Table 1 below. In someembodiments, a disease listed in the table below is treated by atherapeutic polypeptide or therapeutic nucleic acid of the inventionthat complements or otherwise compensates for the correspondingenzymatic defect.

TABLE 1 Lysosomal storage disorders and associated defective enzymes.Lysosomal storage disease Defective enzyme AspartylglusoaminuriaAspartylglucosaminidase Fabry Alpha-galactosidase A Infantile BattenDisease (CNL1) Palmitoyl protein thioesterase Classic Late InfantileTripeptidyl Batten Disease (CNL2) peptidase Juvenile Batten Disease(CNL3) Lysosomal transmembrane protein Batten, other forms multiple gene(CNL4-CNL8) products Cystinosis Cysteine transporter Farber Acidceramidase Fucosidosis Acid alpha-L-fucosidase GalactosidosialidosisProtective protein/cathepsin A Gaucher types 1, 2, and 3 Acidbeta-glucosidase GM1 gangliosidosis Acid beta-galactosidase HunterIduronate-2-sulfatase Hurler-Scheie Alpha-L-iduronidase KrabbeGalactocerebrosidase alpha-mannosidosis Acid alpha-mannosidasebeta-mannosidosis Acid beta-mannosidase Maroteaux-Lamy Arylsulfatase BMetachromatic leukodystrophy Aryl sulfatase A Morquio AN-acetylgalactosamine-6-sulfate Morquio B Acid beta-galactosidaseMucolipidosis II/III N-acetylglucosamine- 1-phosphotransferaseNiemann-Pick A, B Acid sphingomyelinase Niemann-Pick C NPC-1 Pompe acidalpha-glucosidase Sandhoff beta-hexosaminidase B Sanfilippo A HeparanN-sulfatase Sanfilippo B alpha-N-acetylglucosaminidase Sanfilippo CAcetyl-CoA:alpha- glucoasaminide N- acetyltransferase Sanfilippo DN-acetylglucosamine-6-sulfate Schindler diseasealpha-N-acetylgalactosaminidase Schindler-Kanzakialpha-N-acetylgalactosaminidase Sialidosis alpha-neuramidase Slybeta-glucuronidase Tay-Sachs beta-hexosaminidase A Wolman Acid lipase

In some embodiments, the heterologous nucleic acid is operably linked toa promoter. Exemplary promoters include, but are not limited to, thecytomegalovirus (CMV) immediate early promoter, the RSV LTR, the MoMLVLTR, the phosphoglycerate kinase-1 (PGK) promoter, a simian virus 40(SV40) promoter and a CK6 promoter, a transthyretin promoter (TTR), a TKpromoter, a tetracycline responsive promoter (TRE), an HBV promoter, anhAAT promoter, a LSP promoter, chimeric liver-specific promoters (LSPs),the E2F promoter, the telomerase (hTERT) promoter; the cytomegalovirusenhancer/chicken beta-actin/Rabbit 3-globin promoter (CAG promoter; Niwaet al., Gene, 1991, 108(2):193-9) and the elongation factor 1-alphapromoter (EF1-alpha) promoter (Kim et al., Gene, 1990, 91(2):217-23 andGuo et al., Gene Ther., 1996, 3(9):802-10). In some embodiments, thepromoter comprises a human β-glucuronidase promoter or a cytomegalovirusenhancer linked to a chicken β-actin (CBA) promoter. The promoter can bea constitutive, inducible or repressible promoter. In some embodiments,the invention provides a recombinant vector comprising nucleic acidencoding a heterologous transgene of the present disclosure operablylinked to a CBA promoter. Exemplary promoters and descriptions may befound, e.g., in U.S. PG Pub. 20140335054.

Examples of constitutive promoters include, without limitation, theretroviral Rous sarcoma virus (RSV) LTR promoter (optionally with theRSV enhancer), the cytomegalovirus (CMV) promoter (optionally with theCMV enhancer) [see, e.g., Boshart et al., Cell, 41:521-530 (1985)], theSV40 promoter, the dihydrofolate reductase promoter, the 13-actinpromoter, the phosphoglycerol kinase (PGK) promoter, and the EF1apromoter [Invitrogen].

Inducible promoters allow regulation of gene expression and can beregulated by exogenously supplied compounds, environmental factors suchas temperature, or the presence of a specific physiological state, e.g.,acute phase, a particular differentiation state of the cell, or inreplicating cells only. Inducible promoters and inducible systems areavailable from a variety of commercial sources, including, withoutlimitation, Invitrogen, Clontech and Ariad. Many other systems have beendescribed and can be readily selected by one of skill in the art.Examples of inducible promoters regulated by exogenously suppliedpromoters include the zinc-inducible sheep metallothionine (MT)promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus(MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); theecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA,93:3346-3351 (1996)), the tetracycline-repressible system (Gossen etal., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), thetetracycline-inducible system (Gossen et al., Science, 268:1766-1769(1995), see also Harvey et al., Curr. Opin. Chem. Biol., 2:512-518(1998)), the RU486-inducible system (Wang et al., Nat. Biotech.,15:239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997)) and therapamycin-inducible system (Magari et al., J. Clin. Invest.,100:2865-2872 (1997)). Still other types of inducible promoters whichmay be useful in this context are those which are regulated by aspecific physiological state, e.g., temperature, acute phase, aparticular differentiation state of the cell, or in replicating cellsonly.

In another embodiment, the native promoter, or fragment thereof, for thetransgene will be used. The native promoter can be used when it isdesired that expression of the transgene should mimic the nativeexpression. The native promoter may be used when expression of thetransgene must be regulated temporally or developmentally, or in atissue-specific manner, or in response to specific transcriptionalstimuli. In a further embodiment, other native expression controlelements, such as enhancer elements, polyadenylation sites or Kozakconsensus sequences may also be used to mimic the native expression

In some embodiments, the regulatory sequences impart tissue-specificgene expression capabilities. In some cases, the tissue-specificregulatory sequences bind tissue-specific transcription factors thatinduce transcription in a tissue specific manner. Such tissue-specificregulatory sequences (e.g., promoters, enhancers, etc.) are well knownin the art.

In some embodiments, the vector comprises an intron. For example, insome embodiments, the intron is a chimeric intron derived from chickenbeta-actin and rabbit beta-globin. In some embodiments, the intron is aminute virus of mice (MVM) intron.

In some embodiments, the vector comprises a polyadenylation (polyA)sequence. Numerous examples of polyadenylation sequences are known inthe art, such as a bovine growth hormone (BGH) Poly(A) sequence (see,e.g., accession number EF592533), an SV40 polyadenylation sequence, andan HSV TK pA polyadenylation sequence.

V. Viral Particles and Methods of Producing Viral Particles

Certain aspects of the present disclosure relate to recombinant viralparticles (e.g., rAAV particles).

Based on the guidance provided herein, the techniques of the presentdisclosure may suitably be adapted by one of skill in the art for usewith a variety of different viruses.

In some embodiments, the virus is of the family Adenoviridae, whichincludes nonenveloped viruses typically known as Adenoviruses. In someembodiments, the virus is of the genus Atadenovirus, Aviadenovirus,Ichtadenovirus, Mastadenovirus, or Siadenovirus.

In some embodiments, the virus is of the family Parvoviridae, whichincludes nonenveloped viruses such as AAV and Bocaparvovirus. In someembodiments, the virus is of the subfamily Densovirinae. In someembodiments, the virus is of the genus Ambidensovirus, Brevidensovirus,Hepandensovirus, Iteradensovirus, or Penstyldensovirus. In someembodiments, the virus is of the subfamily Parvovirinae. In someembodiments, the virus is of the genus Amdoparvovirus, Aveparvovirus,Bocaparvovirus, Copiparvovirus, Dependoparvovirus, Erythroparvovirus,Protoparvovirus, or Tetraparvovirus.

In some embodiments, the virus is of the family Retroviridae, whichincludes enveloped viruses including lentivirus. In some embodiments,the virus is of the subfamily Orthoretrovirinae. In some embodiments,the virus is of the genus Alpharetrovirus, Betaretrovirus,Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, or Lentivirus. Insome embodiments, the virus is of the subfamily Spumaretrovirinae. Insome embodiments, the virus is of the genus Spumavirus.

In some embodiments, the virus is of the family Baculoviridae, whichincludes enveloped viruses including alphabaculovirus. In someembodiments, the virus is of the genus Alphabaculovirus,Betabaculovirus, Deltabaculovirus, or Gammabaculovirus.

In some embodiments, the virus is of the family Herpesviridae, whichincludes enveloped viruses such as the simplex viruses HSV-1 and HSV-2.In some embodiments, the virus is of the subfamily Alphaherpesvirinae.In some embodiments, the virus is of the genus Iltovirus, Mardivirus,Simplexvirus, or Varicellovirus. In some embodiments, the virus is ofthe subfamily Betaherpesvirinae. In some embodiments, the virus is ofthe genus Cytomegalovirus, Muromegalovirus, Proboscivirus, orRoseolovirus. In some embodiments, the virus is of the subfamilyGammaherpesvirinae. In some embodiments, the virus is of the genusLymphocryptovirus, Macavirus, Percavirus, or Rhadinovirus.

In some embodiments, the virus is an AAV virus. In an AAV particle, anucleic acid is encapsidated in the AAV particle. The AAV particle alsocomprises capsid proteins. In some embodiments, the nucleic acidcomprises a heterologous nucleic acid and/or one or more of thefollowing components, operatively linked in the direction oftranscription, control sequences including transcription initiation andtermination sequences, thereby forming an expression cassette.

In some embodiments, the viral particle comprises an AAV ITR sequence.For example, an expression cassette may be flanked on the 5′ and 3′ endby at least one functional AAV ITR sequence. By “functional AAV ITRsequences” it is meant that the ITR sequences function as intended forthe rescue, replication and packaging of the AAV virion. See Davidson etal., PNAS, 2000, 97(7)3428-32; Passini et al., J. Virol., 2003,77(12):7034-40; and Pechan et al., Gene Ther., 2009, 16:10-16, all ofwhich are incorporated herein in their entirety by reference. Forpracticing some aspects of the invention, the recombinant vectorscomprise at least all of the sequences of AAV essential forencapsidation and the physical structures for infection by the rAAV. AAVITRs for use in the vectors of the invention need not have a wild-typenucleotide sequence (e.g., as described in Kotin, Hum. Gene Ther., 1994,5:793-801), and may be altered by the insertion, deletion orsubstitution of nucleotides or the AAV ITRs may be derived from any ofseveral AAV serotypes. More than 40 serotypes of AAV are currentlyknown, and new serotypes and variants of existing serotypes continue tobe identified. See Gao et al., PNAS, 2002, 99(18): 11854-6; Gao et al.,PNAS, 2003, 100(10):6081-6; and Bossis et al., J. Virol., 2003,77(12):6799-810. Use of any AAV serotype is considered within the scopeof the present invention. In some embodiments, a rAAV vector is a vectorderived from an AAV serotype, including without limitation, AAV1, AAV2,AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10,AAVrh10, AAV11, AAV12, AAV LK03, AAV2R471A, AAV DJ, AAV DJ8, a goat AAV,bovine AAV, or mouse AAV ITRs or the like. In some embodiments, thenucleic acid in the AAV (e.g., an rAAV vector) comprises an ITR of AAV1,AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10,AAVrh10, AAV11, AAV12, AAV LK03, AAV2R471A, AAV DJ, AAV DJ8, a goat AAV,bovine AAV, or mouse AAV ITRs or the like. In some embodiments, the AAVparticle comprises an AAV vector encoding a heterologous transgeneflanked by one or more AAV ITRs.

In some embodiments, a rAAV particle comprises an encapsulation proteinselected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6 (e.g., a wild-type AAV6capsid, or a variant AAV6 capsid such as ShH10, as described in U.S. PGPub. 2012/0164106), AAV7, AAV8, AAVrh8, AAVrh8R, AAV9 (e.g., a wild-typeAAV9 capsid, or a modified AAV9 capsid as described in U.S. PG Pub.2013/0323226), AAV10, AAVrh10, AAV11, AAV12, a tyrosine capsid mutant, aheparin binding capsid mutant, an AAV2R471A capsid, an AAVAAV2/2-7m8capsid, an AAV LK03 capsid, an AAV DJ capsid (e.g., an AAV-DJ/8 capsid,an AAV-DJ/9 capsid, or any other of the capsids described in U.S. PGPub. 2012/0066783), AAV2 N587A capsid, AAV2 E548A capsid, AAV2 N708Acapsid, AAV V708K capsid, goat AAV capsid, AAV1/AAV2 chimeric capsid,bovine AAV capsid, mouse AAV capsid, rAAV2/HBoV1 capsid, an AAV2HBKOcapsid, an AAVPHP.B capsid or an AAVPHP.eB capsid, or an AAV capsiddescribed in U.S. Pat. No. 8,283,151 or International Publication No.WO/2003/042397. In further embodiments, a rAAV particle comprises capsidproteins of an AAV serotype from Clades A-F.

Certain aspects of the present disclosure relate to an AAV (e.g., arAAV) capsid protein comprising an amino acid substitution at amino acidresidue 2. In some embodiments, the amino acid substitution at aminoacid residue 2 alters N-terminal acetylation compared to N-terminalacetylation at amino acid residue 2 of the parent AAV capsid protein. Asdescribed herein, the amino acid at the 2^(nd) position to theinitiating methionine (iMet X) of an AAV capsid protein may be examinedfor effects on N-terminal acetylation, trafficking, transduction, and/orother post-translational modification(s) (e.g., glycosylation,ubiquitination, and so forth). Any assay described herein for examiningacetylation, or a functional consequence thereof related to AAVparticles, may be used to assess N-terminal acetylation. In someembodiments, amino acid residue 2 of the AAV capsid protein (e.g., VP1or VP3) is substituted with Cys, Ser, Thr, Val, Gly, Asn, Asp, Glu, Ile,Leu, Phe, Gln, Lys, Met, Pro or Tyr. In some embodiments, the amino acidsubstitution results in less deamidation of the AAV capsid.

Other aspects of the present disclosure relate to an AAV (e.g., a rAAV)capsid protein comprising an amino acid substitution that altersdeamidation. In some embodiments, an amino acid substitution (e.g., anamino acid substitution at A35 of VP1, N57 of VP1, G58 of VP1, N382 ofVP3, G383 of VP3, N511 of VP3, G512 of VP3, N715 of VP3, or G716 of VP3)that “alters” deamidation results in a higher frequency of deamidationor a lower frequency of deamidation, e.g., as compared to a VP1 or VP3without the substitution, such as the parental VP1 or VP3. As describedherein, a potential deamidation site of an AAV capsid protein (e.g., VP1or VP3) may be examined for effects on deamidation, trafficking,transduction, and/or other post-translational modification(s) (e.g.,glycosylation, ubiquitination, and so forth). Any assay described hereinfor examining deamidation, or a functional consequence thereof relatedto AAV particles, may be used to assess deamidation.

Several potential deamidation sites are described herein. In someembodiments, an amino acid substitution that alters deamidation isselected from A35 of VP1, N57 of VP1, G58 of VP1, N382 of VP3, G383 ofVP3, N511 of VP3, G512 of VP3, N715 of VP3, or G716 of VP3. For example,in some embodiments, N57 of VP1, N382 of VP3, N511 of VP3, and/or N715of VP3 is substituted with Asp, and the amino acid substitution resultsin a higher frequency of deamidation as compared to deamidation of VP1and/or VP3 of the parent AAV particle. In other embodiments, the aminoacid substitution is N57K or N57Q, and the amino acid substitutionresults in a lower frequency of deamidation as compared to deamidationof VP1 and/or VP3 of the parent AAV particle. In yet other embodiments,G58 of VP1, G383 of VP3, G512 of VP3, and/or G716 of VP3 is substitutedwith an amino acid that is not Gly (e.g., Ala, Arg, Asn, Asp, Cys, Glu,Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val), andthe amino acid substitution results in a lower frequency of deamidationas compared to deamidation of VP1 and/or VP3 of the parent AAV particle.

In some embodiments, the AAV capsid protein is VP1, VP2, or VP3. The AAVparticle may comprise any of the exemplary AAV capsid serotypesdescribed herein, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV LK03,AAV2R471A, AAV2/2-7m8, AAV DJ, AAV DJ8, AAV2 N587A, AAV2 E548A, AAV2N708A, AAV V708K, goat AAV, AAV1/AAV2 chimeric, bovine AAV, mouse AAV,or rAAV2/HBoV1. The AAV capsid protein may further comprise any of thecapsid protein mutations described herein, such as tyrosine and/orheparin binding mutations.

Other aspects of the present disclosure relate to methods of improvingthe stability of a rAAV particle. In some embodiments, the methodsinclude substituting amino acid residue 2 of VP1 and/or VP3, e.g., asdescribed herein. For example, in some embodiments, amino acid residue 2of VP1 is substituted. In other embodiments, amino acid residue 2 of VP3is substituted. In some embodiments, the substituted amino acid atposition 2 is N-acetylated at a higher frequency than amino acid residue2 of the parent VP1 and/or VP3, e.g., as described herein. In someembodiments, substituting amino acid residue 2 of VP1 and/or VP3improves the stability of a rAAV particle by at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,or at least about 100%. In some embodiments, the stability of a rAAVparticle with a substituted amino acid at position 2 may be compared toa wild-type or parental AAV capsid, e.g., of the same serotype. Forexample, in some embodiments, substituting amino acid residue 2 of VP1and/or VP3 improves the stability of a rAAV particle by any one of about10% to about 100%, about 20% to about 100%, about 30% to about 100%,about 40% to about 100%, about 50% to about 100%, about 60% to about100%, about 70% to about 100%, about 80% to about 100%, about 90% toabout 100%, about 10% to about 90%, about 20% to about 90%, about 30% toabout 90%, about 40% to about 90%, about 50% to about 90%, about 60% toabout 90%, about 70% to about 90%, about 80% to about 90%, about 10% toabout 80%, about 20% to about 80%, about 30% to about 80%, about 40% toabout 80%, about 50% to about 80%, about 60% to about 80%, about 70% toabout 80%, about 10% to about 70%, about 20% to about 70%, about 30% toabout 70%, about 40% to about 70%, about 50% to about 70%, about 60% toabout 70%, about 10% to about 60%, about 20% to about 60%, about 30% toabout 60%, about 40% to about 60%, about 50% to about 60%, about 10% toabout 50%, about 20% to about 50%, about 30% to about 50%, about 40% toabout 50%, about 10% to about 40%, about 20% to about 40%, about 30% toabout 40%, about 10% to about 30%, about 20% to about 30%, or about 10%to about 20%, e.g., as compared to stability of a rAAV particlecomprising a wild-type capsid. AAV particle stability may be measuredusing various assays known in the art, including without limitationdifferential scanning fluorescence (DSF), differential scanningcalorimetry (DSC), other thermal denaturation assays, susceptibility toproteolysis, imaging or structural analysis to observe denaturation(e.g., using electron microscopy), transduction efficiency or anotherfunctional assay on AAV particle compositions kept for a designated timeinterval at a particular temperature (e.g., room temperature, or 4° C.,for thermal stability) or treated at a particular pH (e.g., pHstability), and the like.

Other aspects of the present disclosure relate to methods of improvingthe assembly of a rAAV particle. In some embodiments, the methodsinclude substituting amino acid residue 2 of VP1 and/or VP3, e.g., asdescribed herein. For example, in some embodiments, amino acid residue 2of VP1 is substituted. In other embodiments, amino acid residue 2 of VP3is substituted. In some embodiments, the substituted amino acid atposition 2 is N-acetylated at a higher frequency than amino acid residue2 of the parent VP1 and/or VP3, e.g., as described herein. In someembodiments, substituting amino acid residue 2 of VP1 and/or VP3improves the assembly of a rAAV particle by at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,or at least about 100%. In some embodiments, the assembly of a rAAVparticle with a substituted amino acid at position 2 may be compared toa wild-type or parental AAV capsid, e.g., of the same serotype. Forexample, in some embodiments, substituting amino acid residue 2 of VP1and/or VP3 improves the assembly of a rAAV particle by any one of about10% to about 100%, about 20% to about 100%, about 30% to about 100%,about 40% to about 100%, about 50% to about 100%, about 60% to about100%, about 70% to about 100%, about 80% to about 100%, about 90% toabout 100%, about 10% to about 90%, about 20% to about 90%, about 30% toabout 90%, about 40% to about 90%, about 50% to about 90%, about 60% toabout 90%, about 70% to about 90%, about 80% to about 90%, about 10% toabout 80%, about 20% to about 80%, about 30% to about 80%, about 40% toabout 80%, about 50% to about 80%, about 60% to about 80%, about 70% toabout 80%, about 10% to about 70%, about 20% to about 70%, about 30% toabout 70%, about 40% to about 70%, about 50% to about 70%, about 60% toabout 70%, about 10% to about 60%, about 20% to about 60%, about 30% toabout 60%, about 40% to about 60%, about 50% to about 60%, about 10% toabout 50%, about 20% to about 50%, about 30% to about 50%, about 40% toabout 50%, about 10% to about 40%, about 20% to about 40%, about 30% toabout 40%, about 10% to about 30%, about 20% to about 30%, or about 10%to about 20%, e.g., as compared to assembly of a rAAV particlecomprising a wild-type capsid. AAV particle assembly may be measuredusing various assays known in the art, including without limitation,measuring particle production amount and/or rate, quantifying capsidproduction (e.g., after purification using any of the methods describedherein), assaying production of complete vectors vs. empty capsids,measuring transduction efficiency, imaging or structural analysis toobserve particle formation (e.g., using electron microscopy), productionof AAV capsid proteins (e.g., as assayed by Western blotting), and thelike.

Other aspects of the present disclosure relate to methods of improvingthe transduction of a rAAV particle. In some embodiments, the methodsinclude substituting amino acid residue 2 of VP1 and/or VP3, e.g., asdescribed herein. For example, in some embodiments, amino acid residue 2of VP1 is substituted. In other embodiments, amino acid residue 2 of VP3is substituted. In some embodiments, the substituted amino acid atposition 2 is N-acetylated at a higher frequency than amino acid residue2 of the parent VP1 and/or VP3, e.g., as described herein. In someembodiments, substituting amino acid residue 2 of VP1 and/or VP3improves the transduction of a rAAV particle by at least about 5%, atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, or at least about 100%. In some embodiments, the transduction of arAAV particle with a substituted amino acid at position 2 may becompared to a wild-type or parental AAV capsid, e.g., of the sameserotype. For example, in some embodiments, substituting amino acidresidue 2 of VP1 and/or VP3 improves the transduction of a rAAV particleby any one of about 10% to about 100%, about 20% to about 100%, about30% to about 100%, about 40% to about 100%, about 50% to about 100%,about 60% to about 100%, about 70% to about 100%, about 80% to about100%, about 90% to about 100%, about 10% to about 90%, about 20% toabout 90%, about 30% to about 90%, about 40% to about 90%, about 50% toabout 90%, about 60% to about 90%, about 70% to about 90%, about 80% toabout 90%, about 10% to about 80%, about 20% to about 80%, about 30% toabout 80%, about 40% to about 80%, about 50% to about 80%, about 60% toabout 80%, about 70% to about 80%, about 10% to about 70%, about 20% toabout 70%, about 30% to about 70%, about 40% to about 70%, about 50% toabout 70%, about 60% to about 70%, about 10% to about 60%, about 20% toabout 60%, about 30% to about 60%, about 40% to about 60%, about 50% toabout 60%, about 10% to about 50%, about 20% to about 50%, about 30% toabout 50%, about 40% to about 50%, about 10% to about 40%, about 20% toabout 40%, about 30% to about 40%, about 10% to about 30%, about 20% toabout 30%, or about 10% to about 20%, e.g., as compared to transductionof a rAAV particle comprising a wild-type capsid. AAV particletransduction may be measured using various assays known in the art,including without limitation, the transduction efficiency assaysdescribed herein. In some embodiments, the invention provide methods ofreducing the transduction of a rAAV particle; for example, bysubstituting amino acid residue 2 of VP1 and/or VP3.

Other aspects of the present disclosure relate to methods of improvingthe stability of a rAAV particle. In some embodiments, the methodsinclude substituting an amino acid of VP1 and/or VP3 that altersdeamidation, e.g., as described herein. For example, in someembodiments, A35 of VP1, N57 of VP1, G58 of VP1, N382 of VP3, G383 ofVP3, N511 of VP3, G512 of VP3, N715 of VP3, or G716 of VP3 issubstituted. In some embodiments, the substituted amino acid isdeamidated at a higher frequency than the amino acid residue of theparent VP1 and/or VP3, e.g., as described herein. In some embodiments,substituting A35 of VP1, N57 of VP1, G58 of VP1, N382 of VP3, G383 ofVP3, N511 of VP3, G512 of VP3, N715 of VP3, and/or G716 of VP3 improvesthe stability of a rAAV particle by at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, or atleast about 100%. In some embodiments, the stability of a rAAV particlewith a substituted A35 of VP1, N57 of VP1, G58 of VP1, N382 of VP3, G383of VP3, N511 of VP3, G512 of VP3, N715 of VP3, or G716 of VP3 may becompared to a wild-type or parental AAV capsid, e.g., of the sameserotype. For example, in some embodiments, substituting A35 of VP1, N57of VP1, G58 of VP1, N382 of VP3, G383 of VP3, N511 of VP3, G512 of VP3,N715 of VP3, and/or G716 of VP3 improves the stability of a rAAVparticle by any one of about 10% to about 100%, about 20% to about 100%,about 30% to about 100%, about 40% to about 100%, about 50% to about100%, about 60% to about 100%, about 70% to about 100%, about 80% toabout 100%, about 90% to about 100%, about 10% to about 90%, about 20%to about 90%, about 30% to about 90%, about 40% to about 90%, about 50%to about 90%, about 60% to about 90%, about 70% to about 90%, about 80%to about 90%, about 10% to about 80%, about 20% to about 80%, about 30%to about 80%, about 40% to about 80%, about 50% to about 80%, about 60%to about 80%, about 70% to about 80%, about 10% to about 70%, about 20%to about 70%, about 30% to about 70%, about 40% to about 70%, about 50%to about 70%, about 60% to about 70%, about 10% to about 60%, about 20%to about 60%, about 30% to about 60%, about 40% to about 60%, about 50%to about 60%, about 10% to about 50%, about 20% to about 50%, about 30%to about 50%, about 40% to about 50%, about 10% to about 40%, about 20%to about 40%, about 30% to about 40%, about 10% to about 30%, about 20%to about 30%, or about 10% to about 20%, e.g., as compared to stabilityof a rAAV particle comprising a wild-type capsid. AAV particle stabilitymay be measured using various assays known in the art, including withoutlimitation differential scanning fluorescence (DSF), differentialscanning calorimetry (DSC), other thermal denaturation assays,susceptibility to proteolysis, imaging or structural analysis to observedenaturation (e.g., using electron microscopy), transduction efficiencyor another functional assay on AAV particle compositions kept for adesignated time interval at a particular temperature (e.g., roomtemperature, or 4° C., for thermal stability) or treated at a particularpH (e.g., pH stability), and the like.

Other aspects of the present disclosure relate to methods of improvingthe assembly of a rAAV particle. In some embodiments, the methodsinclude substituting an amino acid of VP1 and/or VP3 that altersdeamidation, e.g., as described herein. For example, in someembodiments, A35 of VP1, N57 of VP1, G58 of VP1, N382 of VP3, G383 ofVP3, N511 of VP3, G512 of VP3, N715 of VP3, or G716 of VP3 issubstituted. In some embodiments, the substituted amino acid isdeamidated at a higher frequency than the amino acid residue of theparent VP1 and/or VP3, e.g., as described herein. In some embodiments,substituting A35 of VP1, N57 of VP1, G58 of VP1, N382 of VP3, G383 ofVP3, N511 of VP3, G512 of VP3, N715 of VP3, and/or G716 of VP3 improvesthe assembly of a rAAV particle by at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, or atleast about 100%. In some embodiments, the stability of a rAAV particlewith a substituted A35 of VP1, N57 of VP1, G58 of VP1, N382 of VP3, G383of VP3, N511 of VP3, G512 of VP3, N715 of VP3, or G716 of VP3 may becompared to a wild-type or parental AAV capsid, e.g., of the sameserotype. For example, in some embodiments, substituting A35 of VP1, N57of VP1, G58 of VP1, N382 of VP3, G383 of VP3, N511 of VP3, G512 of VP3,N715 of VP3, and/or G716 of VP3 improves the assembly of a rAAV particleby any one of about 10% to about 100%, about 20% to about 100%, about30% to about 100%, about 40% to about 100%, about 50% to about 100%,about 60% to about 100%, about 70% to about 100%, about 80% to about100%, about 90% to about 100%, about 10% to about 90%, about 20% toabout 90%, about 30% to about 90%, about 40% to about 90%, about 50% toabout 90%, about 60% to about 90%, about 70% to about 90%, about 80% toabout 90%, about 10% to about 80%, about 20% to about 80%, about 30% toabout 80%, about 40% to about 80%, about 50% to about 80%, about 60% toabout 80%, about 70% to about 80%, about 10% to about 70%, about 20% toabout 70%, about 30% to about 70%, about 40% to about 70%, about 50% toabout 70%, about 60% to about 70%, about 10% to about 60%, about 20% toabout 60%, about 30% to about 60%, about 40% to about 60%, about 50% toabout 60%, about 10% to about 50%, about 20% to about 50%, about 30% toabout 50%, about 40% to about 50%, about 10% to about 40%, about 20% toabout 40%, about 30% to about 40%, about 10% to about 30%, about 20% toabout 30%, or about 10% to about 20%, e.g., as compared to assembly of arAAV particle comprising a wild-type capsid. AAV particle assembly maybe measured using various assays known in the art, including withoutlimitation, measuring particle production amount and/or rate,quantifying capsid production (e.g., after purification using any of themethods described herein), assaying production of complete vectors vs.empty capsids, measuring transduction efficiency, imaging or structuralanalysis to observe particle formation (e.g., using electronmicroscopy), production of AAV capsid proteins (e.g., as assayed byWestern blotting), and the like.

Other aspects of the present disclosure relate to methods of improvingthe transduction of a rAAV particle. In some embodiments, the methodsinclude substituting an amino acid of VP1 and/or VP3 that altersdeamidation, e.g., as described herein. For example, in someembodiments, A35 of VP1, N57 of VP1, G58 of VP1, N382 of VP3, G383 ofVP3, N511 of VP3, G512 of VP3, N715 of VP3, or G716 of VP3 issubstituted. In some embodiments, the substituted amino acid isdeamidated at a higher frequency than the amino acid residue of theparent VP1 and/or VP3, e.g., as described herein. In some embodiments,substituting A35 of VP1, N57 of VP1, G58 of VP1, N382 of VP3, G383 ofVP3, N511 of VP3, G512 of VP3, N715 of VP3, and/or G716 of VP3 improvesthe transduction of a rAAV particle by at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, or atleast about 100%. In some embodiments, the stability of a rAAV particlewith a substituted A35 of VP1, N57 of VP1, G58 of VP1, N382 of VP3, G383of VP3, N511 of VP3, G512 of VP3, N715 of VP3, or G716 of VP3 may becompared to a wild-type or parental AAV capsid, e.g., of the sameserotype. For example, in some embodiments, substituting A35 of VP1, N57of VP1, G58 of VP1, N382 of VP3, G383 of VP3, N511 of VP3, G512 of VP3,N715 of VP3, and/or G716 of VP3 improves the transduction of a rAAVparticle by any one of about 10% to about 100%, about 20% to about 100%,about 30% to about 100%, about 40% to about 100%, about 50% to about100%, about 60% to about 100%, about 70% to about 100%, about 80% toabout 100%, about 90% to about 100%, about 10% to about 90%, about 20%to about 90%, about 30% to about 90%, about 40% to about 90%, about 50%to about 90%, about 60% to about 90%, about 70% to about 90%, about 80%to about 90%, about 10% to about 80%, about 20% to about 80%, about 30%to about 80%, about 40% to about 80%, about 50% to about 80%, about 60%to about 80%, about 70% to about 80%, about 10% to about 70%, about 20%to about 70%, about 30% to about 70%, about 40% to about 70%, about 50%to about 70%, about 60% to about 70%, about 10% to about 60%, about 20%to about 60%, about 30% to about 60%, about 40% to about 60%, about 50%to about 60%, about 10% to about 50%, about 20% to about 50%, about 30%to about 50%, about 40% to about 50%, about 10% to about 40%, about 20%to about 40%, about 30% to about 40%, about 10% to about 30%, about 20%to about 30%, or about 10% to about 20%, e.g., as compared totransduction of a rAAV particle comprising a wild-type capsid. AAVparticle transduction may be measured using various assays known in theart, including without limitation, the transduction efficiency assaysdescribed herein.

In some aspects, the invention provides viral particles comprising arecombinant self-complementing genome (e.g., a self-complementary orself-complimenting rAAV vector). AAV viral particles withself-complementing vector genomes and methods of use ofself-complementing AAV genomes are described in U.S. Pat. Nos.6,596,535; 7,125,717; 7,465,583; 7,785,888; 7,790,154; 7,846,729;8,093,054; and 8,361,457; and Wang Z., et al., (2003) Gene Ther10:2105-2111, each of which are incorporated herein by reference in itsentirety. A rAAV comprising a self-complementing genome will quicklyform a double stranded DNA molecule by virtue of its partiallycomplementing sequences (e.g., complementing coding and non-codingstrands of a heterologous nucleic acid). In some embodiments, the vectorcomprises a first nucleic acid sequence encoding a heterologous nucleicacid and a second nucleic acid sequence encoding a complement of thenucleic acid, where the first nucleic acid sequence can form intrastrandbase pairs with the second nucleic acid sequence along most or all ofits length.

In some embodiments, the first heterologous nucleic acid sequence and asecond heterologous nucleic acid sequence are linked by a mutated ITR(e.g., the right ITR). In some embodiments, the ITR comprises thepolynucleotide sequence 5′-CACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCACGCCCGGGCTTTGCCCGGGCG-3′ (SEQ ID NO:8). The mutatedITR comprises a deletion of the D region comprising the terminalresolution sequence. As a result, on replicating an AAV viral genome,the rep proteins will not cleave the viral genome at the mutated ITR andas such, a recombinant viral genome comprising the following in 5′ to 3′order will be packaged in a viral capsid: an AAV ITR, the firstheterologous polynucleotide sequence including regulatory sequences, themutated AAV ITR, the second heterologous polynucleotide in reverseorientation to the first heterologous polynucleotide and a third AAVITR.

Different AAV serotypes are used to optimize transduction of particulartarget cells or to target specific cell types within a particular targettissue (e.g., a diseased tissue). A rAAV particle can comprise viralproteins and viral nucleic acids of the same serotype or a mixedserotype. For example, a rAAV particle may contain one or more ITRs andcapsid derived from the same AAV serotype, or a rAAV particle maycontain one or more ITRs derived from a different AAV serotype thancapsid of the rAAV particle.

In some embodiments, the AAV capsid comprises a mutation, e.g., thecapsid comprises a mutant capsid protein. In some embodiments, themutation is a tyrosine mutation or a heparin binding mutation. In someembodiments, a mutant capsid protein maintains the ability to form anAAV capsid. In some embodiments, the rAAV particle comprises an AAV2 orAAV5 tyrosine mutant capsid (see, e.g., Zhong L. et al., (2008) ProcNatl Acad Sci USA 105(22):7827-7832), such as a mutation in Y444 or Y730(numbering according to AAV2). In further embodiments, the rAAV particlecomprises capsid proteins of an AAV serotype from Clades A-F (Gao, etal., J. Virol. 2004, 78(12):6381).

In some embodiments, a capsid protein comprises one or more amino acidsubstitutions at one or more positions that interact with a heparinsulfate proteoglycan or at one or more positions corresponding to aminoacids 484, 487, 527, 532, 585 or 588, numbering based on VP1 numberingof AAV2. Heparan sulfate proteoglycan (HSPG) is known in the art to actas the cellular receptor for AAV2 particles (Summerford, C. andSamulski, R. J. (1998) J. Virol. 72(2):1438-45). Binding between an AAV2particle and HSPG at the cell membrane serves to attach the particle tothe cell. Other cell surface proteins such as fibroblast growth factorreceptor and αvβ5 integrin may also facilitate cellular infection. Afterbinding, an AAV2 particle may enter the cell through mechanismsincluding receptor mediated endocytosis via clathrin-coated pits. AnAAV2 particle may be released from an endocytic vesicle upon endosomalacidification. This allows the AAV2 particle to travel to theperinuclear region and then the cell nucleus. AAV3 particles are alsoknown to bind heparin (Rabinowitz, J. E., et al. (2002) J. Virol.76(2):791-801).

The binding between AAV2 capsid proteins and HSPG is known to occur viaelectrostatic interactions between basic AAV2 capsid protein residuesand negatively charged glycosaminoglycan residues (Opie, S R et al.,(2003) J. Virol. 77:6995-7006; Kern, A et al., (2003) J. Virol.77:11072-11081). Specific capsid residues implicated in theseinteractions include R484, R487, K527, K532, R585, and R588. Mutationsin these residues have been shown to reduce AAV2 binding to Hela cellsand heparin itself (Opie, S R et al., (2003) J. Virol. 77:6995-7006;Kern, A et al., (2003) J. Virol. 77:11072-11081; WO 2004/027019 A2, U.S.Pat. No. 7,629,322). Further, without wishing to be bound to theory, itis thought that amino acid substitution(s) at one or more of theresidues corresponding to amino acids 484, 487, 527, 532, 585 or 588,numbering based on VP1 numbering of AAV2 may modulate the transductionproperties of AAV capsid types that do not bind to HSPG, or may modulatethe transduction properties of AAV capsid types independent from theirability to bind HSPG. In some embodiments, the one or more amino acidsubstitutions comprises a substitution at position R484, R487, K527,K532, R585 and/or R588 of VP1, VP2 and/or VP3, numbering based on VP1 ofAAV2.

In some embodiments, the one or more amino acid substitutions reducebinding of the rAAV particle to the heparin sulfate proteoglycan byabout at least 10%, about at least 25%, about at least 50%, about atleast 75%, or about at least 100%. In some embodiments, the one or moreamino acid substitutions reduce binding of the rAAV particle to theheparin sulfate proteoglycan by about at least 10%, about at least 15%,about at least 20%, about at least 25%, about at least 30%, about atleast 35%, about at least 40%, about at least 45%, about at least 50%,about at least 55%, about at least 60%, about at least 65%, about atleast 70%, about at least 75%, about at least 80%, about at least 85%,about at least 90%, about at least 95%, or about at least 100% (ascompared to binding of a rAAV particle comprising a wild-type capsid).In some embodiments, the one or more amino acid substitutions reducebinding of the rAAV particle to the heparin sulfate proteoglycan by anyone of about 10% to about 100%, about 20% to about 100%, about 30% toabout 100%, about 40% to about 100%, about 50% to about 100%, about 60%to about 100%, about 70% to about 100%, about 80% to about 100%, about90% to about 100%, about 10% to about 90%, about 20% to about 90%, about30% to about 90%, about 40% to about 90%, about 50% to about 90%, about60% to about 90%, about 70% to about 90%, about 80% to about 90%, about10% to about 80%, about 20% to about 80%, about 30% to about 80%, about40% to about 80%, about 50% to about 80%, about 60% to about 80%, about70% to about 80%, about 10% to about 70%, about 20% to about 70%, about30% to about 70%, about 40% to about 70%, about 50% to about 70%, about60% to about 70%, about 10% to about 60%, about 20% to about 60%, about30% to about 60%, about 40% to about 60%, about 50% to about 60%, about10% to about 50%, about 20% to about 50%, about 30% to about 50%, about40% to about 50%, about 10% to about 40%, about 20% to about 40%, about30% to about 40%, about 10% to about 30%, about 20% to about 30%, orabout 10% to about 20%, (as compared to binding of a rAAV particlecomprising a wild-type capsid). In some embodiments, the one or moreamino acid substitutions results in no detectable binding of the rAAVparticle to the heparin sulfate proteoglycan compared to binding of awild-type rAAV particle. Means to measure binding of AAV particles toHSPG are known in the art; e.g., binding to a heparin sulfatechromatography media or binding to a cell known to express HSPG on itssurface. For example, see Opie, S R et al., (2003) J. Virol.77:6995-7006 and Kern, A et al., (2003) J. Virol. 77:11072-11081. Insome embodiments, the one or more amino acid substitutions improve thetransduction efficiency of the rAAV particle to a cell (e.g., a cell inthe eye or CNS) by about at least 10%, about at least 15%, about atleast 20%, about at least 25%, about at least 30%, about at least 35%,about at least 40%, about at least 45%, about at least 50%, about atleast 55%, about at least 60%, about at least 65%, about at least 70%,about at least 75%, about at least 80%, about at least 85%, about atleast 90%, about at least 95%, or about at least 100% (as compared totransduction efficiency of a rAAV particle comprising a wild-typecapsid). In some embodiments, the one or more amino acid substitutionsimprove the transduction efficiency of the rAAV particle to a cell(e.g., a cell in the eye or CNS) by any one of about 10% to about 100%,about 20% to about 100%, about 30% to about 100%, about 40% to about100%, about 50% to about 100%, about 60% to about 100%, about 70% toabout 100%, about 80% to about 100%, about 90% to about 100%, about 10%to about 90%, about 20% to about 90%, about 30% to about 90%, about 40%to about 90%, about 50% to about 90%, about 60% to about 90%, about 70%to about 90%, about 80% to about 90%, about 10% to about 80%, about 20%to about 80%, about 30% to about 80%, about 40% to about 80%, about 50%to about 80%, about 60% to about 80%, about 70% to about 80%, about 10%to about 70%, about 20% to about 70%, about 30% to about 70%, about 40%to about 70%, about 50% to about 70%, about 60% to about 70%, about 10%to about 60%, about 20% to about 60%, about 30% to about 60%, about 40%to about 60%, about 50% to about 60%, about 10% to about 50%, about 20%to about 50%, about 30% to about 50%, about 40% to about 50%, about 10%to about 40%, about 20% to about 40%, about 30% to about 40%, about 10%to about 30%, about 20% to about 30%, or about 10% to about 20%, (ascompared to transduction efficiency of a rAAV particle comprising awild-type capsid). Means to measure transduction efficiency of AAVparticles to a cell (e.g., a cell in culture or part of a tissue) areknown in the art. For example, a population of cells (e.g., in cultureor part of a tissue) may be infected with a concentration of rAAVparticles containing a vector that, when expressed in the cells,produces an assayable reporter (e.g., GFP fluorescence, sFLT production,etc.).

AAV Capsid Proteins

In some aspects, the invention provides an AAV capsid protein comprisingan amino acid substitution at amino acid residue 2; wherein the aminoacid substitution at amino acid residue 2 alters N-terminal acetylationcompared to N-terminal acetylation at amino acid residue 2 of the parentAAV capsid protein. In some embodiments, the AAV capsid protein is VP1or VP3. In some embodiments, amino acid residue 2 of the AAV capsidprotein (e.g., VP1 or VP3) is substituted with Cys, Ser, Thr, Val, Gly,Asn, Asp, Glu, Ile, Leu, Phe, Gln, Lys, Met, Pro or Tyr. In someembodiments, the amino acid substitution results in less deamidation ofthe AAV capsid protein. Non-limiting examples of AAV capsid proteins ofthe invention include VP1 and/or VP3 of any of the following AAVserotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8,AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV LK03, AAV2R471A,AAV2/2-7m8, AAV DJ, AAV DJ8, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAVV708K, goat AAV, AAV1/AAV2 chimeric, bovine AAV, mouse AAV, orrAAV2/HBoV1 serotype capsid. In some embodiments, the AAV capsid furthercomprises a tyrosine mutation or a heparin binding mutation.

Production of AAV Particles

Numerous methods are known in the art for production of rAAV vectors,including transfection, stable cell line production, and infectioushybrid virus production systems which include adenovirus-AAV hybrids,herpesvirus-AAV hybrids (Conway, J E et al., (1997) J. Virology71(11):8780-8789) and baculovirus-AAV hybrids (Urabe, M. et al., (2002)Human Gene Therapy 13(16):1935-1943; Kotin, R. (2011) Hum Mol Genet.20(R1): R2-R6). rAAV production cultures for the production of rAAVviral particles all require; 1) suitable host cells, 2) suitable helpervirus function, 3) AAV rep and cap genes and gene products; 4) a nucleicacid (such as a therapeutic nucleic acid) flanked by at least one AAVITR sequences (e.g., an AAV genome encoding GNPTAB); and 5) suitablemedia and media components to support rAAV production. In someembodiments, the suitable host cell is a primate host cell. In someembodiments, the suitable host cell is a human-derived cell lines suchas HeLa, A549, 293, or Perc.6 cells. In some embodiments, the suitablehelper virus function is provided by wild-type or mutant adenovirus(such as temperature sensitive adenovirus), herpes virus (HSV),baculovirus, or a plasmid construct providing helper functions. In someembodiments, the AAV rep and cap gene products may be from any AAVserotype. In general, but not obligatory, the AAV rep gene product is ofthe same serotype as the ITRs of the rAAV vector genome as long as therep gene products may function to replicated and package the rAAVgenome. Suitable media known in the art may be used for the productionof rAAV vectors. These media include, without limitation, media producedby Hyclone Laboratories and JRH including Modified Eagle Medium (MEM),Dulbecco's Modified Eagle Medium (DMEM), custom formulations such asthose described in U.S. Pat. No. 6,566,118, and Sf-900 II SFM media asdescribed in U.S. Pat. No. 6,723,551, each of which is incorporatedherein by reference in its entirety, particularly with respect to custommedia formulations for use in production of recombinant AAV vectors. Insome embodiments, the AAV helper functions are provided by adenovirus orHSV. In some embodiments, the AAV helper functions are provided bybaculovirus and the host cell is an insect cell (e.g., Spodopterafrugiperda (Sf9) cells). In some embodiments, the AAV cap functionsprovide an amino acid substitution at amino acid residue 2 of VP1 and/orVP3, wherein the amino acid substitution at amino acid residue 2 of VP1and/or VP3 alters N-terminal acetylation compared to N-terminalacetylation at amino acid residue 2 of VP1 and/or VP3 of the parent AAVparticle. In some embodiments, amino acid residue 2 of the AAV capsidprotein (e.g., VP1 or VP3) is substituted with Cys, Ser, Thr, Val, Gly,Asn, Asp, Glu, Ile, Leu, Phe, Gln, Lys, Met, Pro or Tyr. In someembodiments, the amino acid substitution results in less deamidation ofthe AAV capsid.

One method for producing rAAV particles is the triple transfectionmethod. Briefly, a plasmid containing a rep gene and a capsid gene,along with a helper adenoviral plasmid, may be transfected (e.g., usingthe calcium phosphate method) into a cell line (e.g., HEK-293 cells),and virus may be collected and optionally purified. As such, in someembodiments, the rAAV particle was produced by triple transfection of anucleic acid encoding the rAAV vector, a nucleic acid encoding AAV repand cap, and a nucleic acid encoding AAV helper virus functions into ahost cell, wherein the transfection of the nucleic acids to the hostcells generates a host cell capable of producing rAAV particles.

In some embodiments, rAAV particles may be produced by a producer cellline method (see Martin et al., (2013) Human Gene Therapy Methods24:253-269; U.S. PG Pub. No. US2004/0224411; and Liu, X. L. et al.(1999) Gene Ther. 6:293-299). Briefly, a cell line (e.g., a HeLa, 293,A549, or Perc.6 cell line) may be stably transfected with a plasmidcontaining a rep gene, a capsid gene, and a vector genome comprising apromoter-heterologous nucleic acid sequence (e.g., GNPTAB). Cell linesmay be screened to select a lead clone for rAAV production, which maythen be expanded to a production bioreactor and infected with a helpervirus (e.g., an adenovirus or HSV) to initiate rAAV production. Virusmay subsequently be harvested, adenovirus may be inactivated (e.g., byheat) and/or removed, and the rAAV particles may be purified. As such,in some embodiments, the rAAV particle was produced by a producer cellline comprising one or more of nucleic acid encoding the rAAV vector, anucleic acid encoding AAV rep and cap, and a nucleic acid encoding AAVhelper virus functions. As described herein, the producer cell linemethod may be advantageous for the production of rAAV particles with anoversized genome, as compared to the triple transfection method.

In some embodiments, the nucleic acid encoding AAV rep and cap genesand/or the rAAV genome are stably maintained in the producer cell line.In some embodiments, nucleic acid encoding AAV rep and cap genes and/orthe rAAV genome is introduced on one or more plasmids into a cell lineto generate a producer cell line. In some embodiments, the AAV rep, AAVcap, and rAAV genome are introduced into a cell on the same plasmid. Inother embodiments, the AAV rep, AAV cap, and rAAV genome are introducedinto a cell on different plasmids. In some embodiments, a cell linestably transfected with a plasmid maintains the plasmid for multiplepassages of the cell line (e.g., 5, 10, 20, 30, 40, 50 or more than 50passages of the cell). For example, the plasmid(s) may replicate as thecell replicates, or the plasmid(s) may integrate into the cell genome. Avariety of sequences that enable a plasmid to replicate autonomously ina cell (e.g., a human cell) have been identified (see, e.g., Krysan, P.J. et al. (1989) Mol. Cell Biol. 9:1026-1033). In some embodiments, theplasmid(s) may contain a selectable marker (e.g., an antibioticresistance marker) that allows for selection of cells maintaining theplasmid. Selectable markers commonly used in mammalian cells includewithout limitation blasticidin, G418, hygromycin B, zeocin, puromycin,and derivatives thereof. Methods for introducing nucleic acids into acell are known in the art and include without limitation viraltransduction, cationic transfection (e.g., using a cationic polymer suchas DEAE-dextran or a cationic lipid such as lipofectamine), calciumphosphate transfection, microinjection, particle bombardment,electroporation, and nanoparticle transfection (for more details, seee.g., Kim, T. K. and Eberwine, J. H. (2010) Anal. Bioanal. Chem.397:3173-3178).

In some embodiments, the nucleic acid encoding AAV rep and cap genesand/or the rAAV genome are stably integrated into the genome of theproducer cell line. In some embodiments, nucleic acid encoding AAV repand cap genes and/or the rAAV genome is introduced on one or moreplasmids into a cell line to generate a producer cell line. In someembodiments, the AAV rep, AAV cap, and rAAV genome are introduced into acell on the same plasmid. In other embodiments, the AAV rep, AAV cap,and rAAV genome are introduced into a cell on different plasmids. Insome embodiments, the plasmid(s) may contain a selectable marker (e.g.,an antibiotic resistance marker) that allows for selection of cellsmaintaining the plasmid. Methods for stable integration of nucleic acidsinto a variety of host cell lines are known in the art. For example,repeated selection (e.g., through use of a selectable marker) may beused to select for cells that have integrated a nucleic acid containinga selectable marker (and AAV cap and rep genes and/or a rAAV genome). Inother embodiments, nucleic acids may be integrated in a site-specificmanner into a cell line to generate a producer cell line. Severalsite-specific recombination systems are known in the art, such asFLP/FRT (see, e.g., O'Gorman, S. et al. (1991) Science 251:1351-1355),Cre/loxP (see, e.g., Sauer, B. and Henderson, N. (1988) Proc. Natl.Acad. Sci. 85:5166-5170), and phi C31-att (see, e.g., Groth, A. C. etal. (2000) Proc. Natl. Acad. Sci. 97:5995-6000).

In some embodiments, the producer cell line is derived from a primatecell line (e.g., a non-human primate cell line, such as a Vero or FRhL-2cell line). In some embodiments, the cell line is derived from a humancell line. In some embodiments, the producer cell line is derived fromHeLa, 293, A549, or PERC.6® (Crucell) cells. For example, prior tointroduction and/or stable maintenance/integration of nucleic acidencoding AAV rep and cap genes and/or the oversized rAAV genome into acell line to generate a producer cell line, the cell line is a HeLa,293, A549, or PERC.6® (Crucell) cell line, or a derivative thereof.

In some embodiments, the producer cell line is adapted for growth insuspension. As is known in the art, anchorage-dependent cells aretypically not able to grow in suspension without a substrate, such asmicrocarrier beads. Adapting a cell line to grow in suspension mayinclude, for example, growing the cell line in a spinner culture with astirring paddle, using a culture medium that lacks calcium and magnesiumions to prevent clumping (and optionally an antifoaming agent), using aculture vessel coated with a siliconizing compound, and selecting cellsin the culture (rather than in large clumps or on the sides of thevessel) at each passage. For further description, see, e.g., ATCCfrequently asked questions document (available on the world wide web atatcc.org/Global/FAQs/9/1/Adapting%20a%20monolayer%20cell%20line%20to%20suspension-40.aspx)and references cited therein.

Suitable AAV production culture media of the present invention may besupplemented with serum or serum-derived recombinant proteins at a levelof 0.5%-20% (v/v or w/v). Alternatively, as is known in the art, AAVvectors may be produced in serum-free conditions which may also bereferred to as media with no animal-derived products. One of ordinaryskill in the art may appreciate that commercial or custom media designedto support production of AAV vectors may also be supplemented with oneor more cell culture components know in the art, including withoutlimitation glucose, vitamins, amino acids, and or growth factors, inorder to increase the titer of AAV in production cultures.

AAV production cultures can be grown under a variety of conditions (overa wide temperature range, for varying lengths of time, and the like)suitable to the particular host cell being utilized. As is known in theart, AAV production cultures include attachment-dependent cultures whichcan be cultured in suitable attachment-dependent vessels such as, forexample, roller bottles, hollow fiber filters, microcarriers, andpacked-bed or fluidized-bed bioreactors. AAV vector production culturesmay also include suspension-adapted host cells such as HeLa, 293, andSF-9 cells which can be cultured in a variety of ways including, forexample, spinner flasks, stirred tank bioreactors, and disposablesystems such as the Wave bag system.

AAV vector particles of the invention may be harvested from AAVproduction cultures by lysis of the host cells of the production cultureor by harvest of the spent media from the production culture, providedthe cells are cultured under conditions known in the art to causerelease of AAV particles into the media from intact cells, as describedmore fully in U.S. Pat. No. 6,566,118). Suitable methods of lysing cellsare also known in the art and include for example multiple freeze/thawcycles, sonication, microfluidization, and treatment with chemicals,such as detergents and/or proteases.

In a further embodiment, the AAV particles are purified. The term“purified” as used herein includes a preparation of AAV particles devoidof at least some of the other components that may also be present wherethe AAV particles naturally occur or are initially prepared from. Thus,for example, isolated AAV particles may be prepared using a purificationtechnique to enrich it from a source mixture, such as a culture lysateor production culture supernatant. Enrichment can be measured in avariety of ways, such as, for example, by the proportion ofDNase-resistant particles (DRPs) or genome copies (gc) present in asolution, or by infectivity, or it can be measured in relation to asecond, potentially interfering substance present in the source mixture,such as contaminants, including production culture contaminants orin-process contaminants, including helper virus, media components, andthe like.

In some embodiments, the AAV production culture harvest is clarified toremove host cell debris. In some embodiments, the production cultureharvest is clarified by filtration through a series of depth filtersincluding, for example, a grade DOHC Millipore Millistak+HC Pod Filter,a grade A1HC Millipore Millistak+HC Pod Filter, and a 0.2 μm FilterOpticap XL1O Millipore Express SHC Hydrophilic Membrane filter.Clarification can also be achieved by a variety of other standardtechniques known in the art, such as, centrifugation or filtrationthrough any cellulose acetate filter of 0.2 μm or greater pore sizeknown in the art.

In some embodiments, the AAV production culture harvest is furthertreated with Benzonase® to digest any high molecular weight DNA presentin the production culture. In some embodiments, the Benzonase® digestionis performed under standard conditions known in the art including, forexample, a final concentration of 1-2.5 units/ml of Benzonase® at atemperature ranging from ambient to 37° C. for a period of 30 minutes toseveral hours.

AAV particles may be isolated or purified using one or more of thefollowing purification steps: equilibrium centrifugation; flow-throughanionic exchange filtration; tangential flow filtration (TFF) forconcentrating the AAV particles; AAV capture by apatite chromatography;heat inactivation of helper virus; AAV capture by hydrophobicinteraction chromatography; buffer exchange by size exclusionchromatography (SEC); nanofiltration; and AAV capture by anionicexchange chromatography, cationic exchange chromatography, or affinitychromatography. These steps may be used alone, in various combinations,or in different orders. In some embodiments, the method comprises allthe steps in the order as described below. Methods to purify AAVparticles are found, for example, in Xiao et al., (1998) Journal ofVirology 72:2224-2232; U.S. Pat. Nos. 6,989,264 and 8,137,948; and WO2010/148143.

Pharmaceutical Compositions

In some embodiments, an AAV particle of the present disclosure (e.g., arAAV particle) is in a pharmaceutical composition. The pharmaceuticalcompositions may be suitable for any mode of administration describedherein or known in the art. In some embodiments, the pharmaceuticalcomposition comprises rAAV particles modified to improve the stabilityand/or improve the transduction efficiency of rAAV particles; forexample, for use in substituting the amino acid residue at position 2 ofVP1 and/or VP3 to improve acetylation of rAAV capsid proteins. In someembodiments, the pharmaceutical composition comprises rAAV particlesmodified to modulate the stability and/or the transduction efficiency ofrAAV particles (e.g., increase stability and/or transduction efficiencyor decrease stability and/or transduction efficiency); for example, foruse in substituting the amino acid residues that modulate deamidation(e.g., increase deamidation or decrease deamidation).

In some embodiments, the rAAV particle is in a pharmaceuticalcomposition comprising a pharmaceutically acceptable excipient. As iswell known in the art, pharmaceutically acceptable excipients arerelatively inert substances that facilitate administration of apharmacologically effective substance and can be supplied as liquidsolutions or suspensions, as emulsions, or as solid forms suitable fordissolution or suspension in liquid prior to use. For example, anexcipient can give form or consistency, or act as a diluent. Suitableexcipients include but are not limited to stabilizing agents, wettingand emulsifying agents, salts for varying osmolarity, encapsulatingagents, pH buffering substances, and buffers. Such excipients includeany pharmaceutical agent suitable for direct delivery to the eye whichmay be administered without undue toxicity. Pharmaceutically acceptableexcipients include, but are not limited to, sorbitol, any of the variousTWEEN compounds, and liquids such as water, saline, glycerol andethanol. Pharmaceutically acceptable salts can be included therein, forexample, mineral acid salts such as hydrochlorides, hydrobromides,phosphates, sulfates, and the like; and the salts of organic acids suchas acetates, propionates, malonates, benzoates, and the like. A thoroughdiscussion of pharmaceutically acceptable excipients is available inREMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991). In someembodiments, the pharmaceutical composition comprising a rAAV particledescribed herein and a pharmaceutically acceptable carrier is suitablefor administration to human. Such carriers are well known in the art(see, e.g., Remington's Pharmaceutical Sciences, 15th Edition, pp.1035-1038 and 1570-1580).

Such pharmaceutically acceptable carriers can be sterile liquids, suchas water and oil, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, and thelike. Saline solutions and aqueous dextrose, polyethylene glycol (PEG)and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. The pharmaceutical compositionmay further comprise additional ingredients, for example preservatives,buffers, tonicity agents, antioxidants and stabilizers, nonionic wettingor clarifying agents, viscosity-increasing agents, and the like. Thepharmaceutical compositions described herein can be packaged in singleunit dosages or in multidosage forms. The compositions are generallyformulated as sterile and substantially isotonic solution.

Kits and Articles of Manufacture

The present invention also provides kits or articles of manufacturecomprising any of the rAAV particles and/or pharmaceutical compositionsof the present disclosure. The kits or articles of manufacture maycomprise any of the rAAV particles or rAAV particle compositions of theinvention. In some embodiments the kits are used to improve thestability and/or improve the transduction efficiency of rAAV particles;for example, for use in substituting the amino acid residue at position2 of VP1 and/or VP3 to improve acetylation of rAAV capsid proteins. Insome embodiments the kits are used to modulate the stability and/or thetransduction efficiency of rAAV particles (e.g., increase stabilityand/or transduction efficiency or decrease stability and/or transductionefficiency); for example, for use in substituting the amino acidresidues that modulate deamidation (e.g., increase deamidation ordecrease deamidation).

In some embodiments, the kits or articles of manufacture further includeinstructions for administration of a composition of rAAV particles. Thekits or articles of manufacture described herein may further includeother materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, syringes, andpackage inserts with instructions for performing any methods describedherein. Suitable packaging materials may also be included and may be anypackaging materials known in the art, including, for example, vials(such as sealed vials), vessels, ampules, bottles, jars, flexiblepackaging (e.g., sealed Mylar or plastic bags), and the like. Thesearticles of manufacture may further be sterilized and/or sealed.

In some embodiments, the kits or articles of manufacture further containone or more of the buffers and/or pharmaceutically acceptable excipientsdescribed herein (e.g., as described in REMINGTON'S PHARMACEUTICALSCIENCES (Mack Pub. Co., N.J. 1991). In some embodiments, the kits orarticles of manufacture include one or more pharmaceutically acceptableexcipients, carriers, solutions, and/or additional ingredients describedherein. The kits or articles of manufacture described herein can bepackaged in single unit dosages or in multidosage forms. The contents ofthe kits or articles of manufacture are generally formulated as sterileand can be lyophilized or provided as a substantially isotonic solution.

EXAMPLES

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. It is understood that the examples andembodiments described herein are for illustrative purposes only and thatvarious modifications or changes in light thereof will be suggested topersons skilled in the art and are to be included within the spirit andpurview of this application and scope of the appended claims.

Example 1: Direct LC/NIS and LC/NIS/NIS for Complete Characterization ofRecombinant AAV Viral Capsid Protein

Recombinant adeno-associated viruses (rAAVs) have become popular genetherapy vectors due to their nonpathogenic nature, ability to infectboth dividing and non-dividing cells and long term gene expression.Currently, AAV-based gene therapies are used in clinical trials fornumerous disease targets, such as muscular dystrophy, hemophilia,Parkinson's disease, Leber's congenital amaurosis and maculardegeneration.

AAV is a small and nonenveloped parvovirus with a single stranded DNAgenome encapsulated in an icosahedra shell. Each capsid includes sixtycopies of three viral capsid proteins VP1 (87 kDa), VP2 (73 kDa) and VP3(62 kDa) in an approximately 1:1:10 ratio. The three viral capsidproteins are expressed from the same open reading frame by usingalternative splicing and an atypical start codon and thus haveoverlapping sequences. VP1 has ˜137 additional N-terminal amino acidresidues compared to VP3 while VP2 has ˜65 additional N-terminal aminoacid residues compared to VP3. At least 13 AAV serotypes and ˜150 genesequences have been isolated from human and non-human primate tissues;AAV serotypes differ in the amino acid sequence of viral capsid proteinsand their corresponding cellular receptors and co-receptors fortargeting.

The AAV capsid, in addition to protecting the genome inside, plays animportant role in mediating receptor binding, escape of virus fromendosome, and transport of viral DNA into nucleus in the viral infectioncycle, thus directly impacting viral infectivity. It has been shown thatthe VP1 N-terminus contains a phospholipase PLA2 domain (a.a. 52-97)which is critical in endosomal escaping of virus [1-3]. N-termini of VP1and VP2 also contain three basic amino acid clusters as nuclearlocalization signals. These sequences are highly conserved amongdifferent AAV serotypes. Mutations of these amino acids have been shownto reduce or abolish infectivity completely [4]. In addition, each AAVserotype has corresponding sequence-specific receptors and co-receptors.For example, heparin sulfate proteoglycan was identified as a majorreceptor of AAV2 and several other co-receptors, including αVβ5integrin, fibroblast growth factor receptor 1, and hepatocyte growthreceptor have been identified [5-8]. Mutation analysis of AAV2 capsidproteins has identified a group of basic amino acids (Arginine484, 487,585, and Lysine532) as a heparin-binding motif which contributes to theheparin and HeLa cell binding [9]. NGR domain in AAV2 was identified asan integrin α5β1 binding domain which is essential for viral cell entry[10]. In summary, viral capsid protein sequences are important incellular targeting and trafficking in the viral infection cycle. Sincedifferent production conditions may cause different expression levels ofviral capsid proteins, post-translational modifications, andtruncations, the viral capsid proteins need to be characterized andmonitored to ensure the product consistency in gene therapy developmentprograms.

Traditionally, SDS-PAGE has been used to characterize the AAV viralcapsid proteins, providing rough molecular weight information such as 87kDa, 73 kDa and 62 kDa. No sequence information was obtained from Edmansequencing, possibly due to the blocked N-termini of viral capsidproteins, except VP2. Although X-ray structures of multiple AAVs havebeen solved, only the VP3 region sequence was observed in the crystalstructures. Fifteen N-terminal amino acid residues of VP3 were stillmissing in the X-ray structure, possibly due to its intrinsic disorder[11-13]. It is possible that the lack of information of VP1 and VP2N-terminal regions in the atomic structure might be due to lowstoichiometry of VP1 and VP2 in the capsid. In addition, N-termini ofVP1 and VP2 are buried inside the capsid and are not accessible toantibodies in the native state as reported in the some literature [3,14, 15]. Conventionally, a Gel-LC/MS method (SDS-PAGE, in-gel trypticdigestion and LC/MS/MS) was used in characterization of VPs [16-18].However, N-termini of VP1, VP2 and VP3 have not been confirmed usingthis approach, since this method failed to obtain 100% sequence coverageof VPs due to the limited recovery of peptide from gel.

Direct analysis using MALDI-TOF MS was reported for several virus capsidproteins including tobacco mosaic virus U2 after dissociation withorganic acid [19]. Direct peptide mapping after amide hydrogen exchangeand mass spectrometry have been used to study the pH-induced structuralchanges in the capsid of brome mosaic virus (BMV) [20]. Since AAVs arenonenveloped viruses containing only capsid proteins and genome, AAVscapsids could be directly analyzed by RP-LC/MS of proteins and LC/MS/MSof peptide mapping to achieve 100% sequence coverage after capsiddissociation without SDS-PAGE separation. The DNA fragments could elutein the void volume and thus have no interference on protein/peptidedetection by LC/MS. In order to investigate these methods, direct LC/MSof different types of AAVs after denaturation was used to monitor theprotein sequence and post-translational modifications of AAV capsidproteins. As described herein, N-termini of VP1, VP2 and VP3 of AAVshave been confirmed by mass spectrometry. Acetylations of N-termini ofVP1 and VP3 were also identified in the different serotype of AAVs.Direct LC/MS/MS peptide mapping of AAVs has also been developed toprovide sequence coverage of VP1, VP2 and VP3 and confirm the N-terminiacetylation of VP1 and VP3.

Methods

Materials and Reagents

Dithiothreitol (DTT), 4-vinylpyridine, ultra-pure formic acid, aceticacid, guanidine-HCl, Tris-HCl and Tris base were purchased from SigmaChemicals (St. Louis, Mo.). Amicon ultra-4 filters were purchased fromMillipore (Billerica, Mass.). The porcine sequencing grade trypsin waspurchased from Promega (Milwaukee, Wis.). Endoproteinase Lys-C and Asp-Nwere purchased from Roche (Germany). Slide-A-Lyzer cassettes with 10,000MWCO were purchased from Pierce (Rockford, Ill.).

Vector Production and Purification

AAV vectors were produced using the transient triple transfection methodas previously described (Xiao, 1998 #123). Briefly, HEK293 cells weretransfected using polyethyleneimine, PEI, and a 1:1:1 ratio of threeplasmids (ITR vector, AAV rep/cap and Ad helper plasmid). The vectorplasmid contains the vector genome CBA-EGFP and ITR sequences from AAV2.EGFP expression is driven by the CMV enhancer chicken beta actin hybridpromoter (CBA) as described (Miyazaki, 1989 #124) (Niwa, 1991 #125). TheAAV rep/cap helpers contained rep sequences from AAV2 and serotypespecific capsid sequences with the nomenclature, rep2/cap2, rep2/cap5,rep2/cap7 etc. The pAd helper used was pHelper (Stratagene/AgilentTechnologies, Santa Clara, Calif.). Purification of AAV was performed asdescribed by Qu et al. (2007, J. Virol. Methods 140:183-192).

LC/MS Intact Protein Analysis

The AAV virions were concentrated with an Amicon ultra-4 filter (10 kDaMWCO) and denatured with 10% acetic acid followed by direct analysis inan Acquity UPLC-Xevo® QTOF MS instrument (Waters, Milford, Mass.). Theseparations were performed with a UPLC BEH C4 or C8 column (1.7 μm, 2.1mm i.d.) at a 0.25 ml/min flow rate. Mobile phase A was 0.1% formic acidin water while mobile phase B was 0.1% formic acid in acetonitrile. Thefinal gradient was as follows: from 10% B to 20% B for 6 minutes, from20% B to 30% B in 10 min, then from 30% to 38% B for 40 minutes. For MSthe capillary voltage and sampling cone voltage were set at 3.5 kV and45 V respectively. The mass spectra were acquired in the sensitivitymode with m/z range of 500-4000. Assisted calibration with sodium iodideas calibrant was performed for mass calibration. MaxEnt1 in Masslynxsoftware was used for protein deconvolution.

Enzymatic Digestions of AAV2 VPs

The concentrated AAV2 virions were denatured with 6 M Guanidine-HCl, 0.1M Tris at pH 8.5. The proteins were reduced with 30 mM DTT at 55° C. for1 hour in darkness and alkylated with 0.07% 4-vinylpyridine at roomtemperature for 2 hours. The reactions were quenched by the addition of1M DTT. The samples were dialyzed with Slide-A-Lyzer cassettes (10,000MWCO) against 25 mM Tris buffer at pH 8.5 for ˜18 hours. After dialysis,the samples were split into three aliquots. Each aliquot was digestedwith trypsin at 1:25 or Lys-C at 1:50 or Asp-N at 1:100 enzyme: proteinratio (wt/wt) for 18 hours at 37° C., respectively.

LC/MS/MS Peptide Mapping

Nano LC/MS/MS was performed in using a NanoAcquity HPLC system (Waters,Milford, Mass.) in conjunction with an Orbitrap Velos mass spectrometer(Thermo-Fisher Scientific, Waltham, Mass.) using home packed nanoLCcolumn (75 μm×10 mm) with Magic C18 with packing material (5 Bruker,Billerica, Mass.) at a 300 nl/min flow rate. The mobile phases A and Bwere 0.1% formic acid in water and acetonitrile, respectively. Thegradient was from 2% B to 60% B in 121 min.

The source parameters for velos were as follows: source voltage: 2.5 kv,capillary temperature 275° C.; S-lens RF level: 55%. Data were acquiredusing the top-ten data dependent method with accurate ms at 60,000resolution and 10 MS/MS in ion trap. Mascot was used for databasesearching against AAV2 viral capsid protein sequences. MS tolerance of10 ppm and ms/ms tolerance of 0.8 Da were used for the database search.

UPLC/MS/MS Peptide Mapping

The protein digests were also analyzed by UPLC/MS/MS in AcquityUPLC-Xevo qTOF MS. A BEH300 C18 column (2.1×150 mm) was used forseparation in the mobile phases with 0.1% formic acid inwater/acetonitrile gradient at a flow rate 0.25 ml/min. The mass spectrawere acquired in the positive MSe mode in the mass range of 200-2000.

Results

AAV Denaturation Method

AAVs can be denatured through a number of methods using detergent, heat,high salt, or buffer with low or high pHs. Heat denaturation can lead toprotein precipitation and as a result reverse phase columns are easilyclogged and over pressurized. Denaturation with high salt requires anadditional desalting step before LC/MS analysis. Denaturing with 10%acetic acid was used for the LC/MS intact protein analysis, as itallowed for clean mass spectrum. For peptide mapping, either 0.1%RapiGest or 6 M Guanidine HCl can be used as a denaturing reagent.

Intact Protein Analysis Method Development

Initial intact protein analysis of AAV2 was performed using an UPLC BEHC4 column at fast gradient. Under this condition, only one single peakin the total ion chromatogram was observed, with a mass corresponding toVP3 (FIG. 1A). Without wishing to be bound by theory, it is thought thatthe absence of VP1 and VP2 is possibly due to low stoichiometry of VP1and VP2 or suppression of VP1 and VP2 signals by VP3 if all VPsco-elute. Increasing injection or column length, using a shallowergradient, and using alternative columns have been attempted in order todetect VP1 and VP2. Higher loading (1.7 μg) with a shallower gradient at0.5% B/min resulted in a shoulder peak on the left (FIG. 1B). Theincrease in column length from 10 cm to 15 cm did not enhance theseparation of the shoulder peak (FIG. 1C). However, the shoulder peakwas further separated from the main peak using a BEH C8 column, withimproved signal intensities observed (FIG. 1D).

As a result, the VP1 and VP2 masses were obtained in this shoulder peakat the signal intensities shown in FIG. 2A. The masses of VP1 and VP3correspond to a.a. 2-735 (acetylation) and a.a. 204-735 (acetylation),respectively (FIGS. 2A&2B). No acetylation was observed in VP2(a.a.139-735). In addition, a minor peak with a smaller mass than VP3was observed, with a mass corresponding to amino acid sequence 212-735with one acetylation (FIG. 2B). These data are consistent with DNAsequences since VP3 contains two ATG initiation codons in AAV2:

(SEQ ID NO:1), resulting in two possible N termini (underlined):MATGSGAPMAD (SEQ ID NO:2). The N-terminal methionine residues were notpresent in both VP1 and VP3 as measured by intact protein analysis. Theacetylation of VP1 and VP3 is not a method-induced artifact(denaturation of AAV by 10% acetic acid) since acetylation of VP1 andVP3 is also observed in an AAV preparation using an alternative denaturemethod without acetic acid. The intact protein data also confirmed thatno glycosylation was present in the viral capsid proteins, even thoughseveral N-linked consensus sequences are present [16].

LC/MS/MS Peptide Mapping

To further confirm the N-termini and acetylation observed in the intactprotein analysis, peptide mapping was performed using multiple enzymesand analyzed using multiple instruments. Various sample preparationmethods, including denaturation methods and desalting steps, have beenevaluated. The final digestion method, including denaturation with 6Mguanidine HCl, reduction and alkylation with 4-vinylpyridine, anddialysis using slide-A-lyzer followed by enzymatic digestion, createdclean peptide mapping with low artificial modifications during thedigestion process. As low as 5 μg starting material was tested, yieldingcomplete sequence coverage using nano LC/MS/MS and UPLC/MS/MS.

Mascot search of tryptic digests from nano LC/MS/MS alone yielded 78%sequence coverage with an ion score 13 cut off as shown in FIG. 3 . Thetwo large missing tryptic peptides, T27 and T38 (boxed) from nanoLC/MS/MS were found in the LC/MS in Xevo TOF MS with BEH C18 column(FIG. 3 ). In addition, most of the T27 and T38 peptide sequences werefurther confirmed by nano LC/MS/MS of Asp-N digests as shown in Italicsin FIG. 3 . The complete N-terminal and C-terminal peptides were coveredby Lys-C digests as underlined in FIG. 3 . Therefore, 100% sequencecoverage of VP1 was achieved through multiple enzyme digestions and twoLC/MS/MS methods.

LC/MS/MS confirmed the N- and C-termini of VP1, VP2 and VP3 andN-terminal acetylation of VP1 and VP3 observed in the intact proteinanalysis. FIGS. 4A-4C show the MS/MS spectra of the VP1 N-terminaltryptic peptide A(Ac)ADGYLPDWLEDTLSEGIR (SEQ ID NO:4) (FIG. 4A), VP2N-terminal Asp-N derived peptide (APGKKRPVEHSPVEP) (SEQ ID NO:15) (FIG.4B), and VP3 N-terminal Asp-N peptide A(Ac)TGSGAPM (SEQ ID NO:5) (FIG.4C). MS/MS has confirmed the location of acetylation at the N-terminalalanine residues in both VP1 and VP3 peptides. The presence ofunmodified y18 and y17 ions, and all detected b ions with 42 Da massshift in FIG. 4A indicates the 42 da-modification is located inN-terminal of VP1. Similarly, the presence of unmodified y3 to y8 ionsin FIG. 4C confirmed the location of acetylation at the N-terminalalanine residue.

Comparison of AAV VP N-Termini

In addition to AAV2, AAV1, AAV5, AAV7, AAV9 and AAV Rh10 have also beenanalyzed by intact protein analysis. The theoretical and predictedmasses of VPs in AAVs are shown in Table 2.

TABLE 2 Theoretical Mass vs Experimental Mass for AAV VPs PredictedActual amino amino Theo- Experi- acid acid retical mental SerotypeIsoform sequence sequence Ms.(Da) Ms.(Da) AAV1 VP1  1-736  2(ac)-73681286 81291 VP2 138-736    139-736 66093 66098 VP3 203-736 204(ac)-73659517 59520 AAV2 VP1  1-735  2(ac)-735 81856 81856 VP2 138-735   139-735 66488 66488 VP3 203-735 204(ac)-735 59974 59974 AAV5 VP1 1-724  2(ac)-724 80336 80336 VP2 137-724    138-724 65283 65284 VP3193-724 194(ac)-724 59463 59463 AAV7 VP1  1-737  2(ac)-737 81564 81567VP2 138-737    139-737 66372 66374 VP3 204-737 213(ac)-737 59101 59103AAV9 VP1  1-736  2(ac)-736 81291 81288 VP2 138-736    139-736 6621066209 VP3 203-736 204(ac)-736 59733 59733 AAVRh10 VP1  1-738  2(ac)-73881455 81455 VP2 138-738    139-738 66253 66252 VP3 204-738 205(ac)-73859634 59634

N-termini, as well as their posttranslational modifications, are highlyconserved among the AAV serotypes analyzed, even though AAV5 is reportedas the most diverse AAV serotype sequence, as shown in the sequencealignments in FIG. 5 . In 11 out of 13 AAV serotypes, the N-termini ofVP1 share an identical 13 amino acid residue sequence (MAADGYLPDWLED)(SEQ ID NO:6) while all 13 AAV serotypes have identical TAP . . .N-terminal sequences in VP2 (FIG. 5 ). LC/MS of AAV2 indicated that T ismissing in VP2 at protein level. The N-termini of VP3 are the mostdiverse among the three viral capsid proteins, with 8 out of 13 AAVserotypes sharing a MA . . . N-terminal sequence. Similar to AAV2, AAV1and AAV Rh10 also have two ATG initiation codons with the first one aspredominant N-terminal based on LC/MS intact protein analysis.Interestingly, though AAV7 has two potential initiation codons(GTGGCTGCAGGCGGTGGCGCACCAATGGCAGACAATAAC . . . ) (SEQ ID NO:7), thesecond initiation codon (ATG) was favorable based on the intact proteinanalysis: the VP3′ with 213(ac)-737 was a predominant peak while VP3with 203(ac)-737 was a minor peak.

Conclusions

Applications of LC/MS Intact Protein Analysis and LC/MS/MS PeptideMapping of AAV VPs in Gene Therapy Research and Development

These results demonstrate that direct LC/MS of different types of AAVsafter denaturation was proved to be a simple and effective way tomonitor the protein sequence and post-translational modifications withaccurate mass measurement in the intact protein level. N-termini of VP1,VP2 and VP3 of AAVs were confirmed by mass spectrometry. Acetylations ofN-termini of VP1 and VP3 were also identified in different serotypes ofAAVs. Direct LC/MS/MS peptide mapping of AAVs was developed, provided100% sequence coverage of VP1, VP2 and VP3, and confirmed the N-terminiacetylation of VPs. The theoretical masses of predicted sequences of 13AAV serotypes based on sequence alignment and intact protein analysis ofseveral AAV serotypes are shown in Table 3.

TABLE 3 Predicted Sequences and Masses Predicted VP1 Predicted VP2Predicted VP3 sequence Mass(Da) sequence Mass(Da) sequence Mass(Da) AAV12(ac)-736 81286 139-736 66093 204(ac)-736 59517 AAV2 2(ac)-735 81856139-735 66488 204(ac)-735 59974 AAV3 2(ac)-736 81571 139-736 66319204(ac)-736 59849 AAV4 2(ac)-734 80550 138-734 65626 198(ac)-734 59529AAV5 2(ac)-724 80336 138-724 65283 194(ac)-724 59463 AAV6 2(ac)-73681322 139-736 66096 204(ac)-736 59519 AAV7 2(ac)-737 81564 139-737 66372213(ac)-737 59101 AAV8 2(ac)-738 81667 139-738 66519 205(ac)-738 59805AAV9 2(ac)-736 81291 139-736 66210 204(ac)-736 59733 AAV10 2(ac)-73881477 139-738 66271 205(ac)-738 59638 AAV11 2(ac)-733 80987 139-73365794 198(ac)-733 59696 AAV12 2(ac)-742 82106 139-742 66905 207(ac)-74259846 AAVRh10 2(ac)-738 81455 139-738 66253 205(ac)-738 59634

The accurate masses of VP1, VP2 and VP3 of each serotype are unique andtherefore intact protein analysis can be used as an identity test todifferentiate AAV capsid serotypes. Tables 4-6 show the mass differencesof VPs among 13 common AAV serotypes. Shown in regular font are deltamasses larger than 10, with delta masses less than 10 bolded.

TABLE 4 Mass Differences of VP1 Among 13 AAV Isotypes AAV1 AAV2 570 AAV2AAV3 285 285 AAV3 AAV4 736 1306 1021 AAV4 AAV5 950 1520 1235 215 AAV5AAV6 36 534 249 772 987 AAV6 AAV7 277 292 8 1013 1228 241 AAV7 AAV8 381189 96 1117 1332 345 104 AAV8 AAV9 5 585 280 741 955 31 272 376 AAV9AAV10 191 379 94 927 1142 155 86 190 186 AAV10 AAV11 299 869 584 436 651335 577 681 304 490 AAV11 AAV12 820 250 535 1555 1770 784 542 439 815629 1119 AAV12 AAVRh10 169 401 116 905 1119 133 109 212 164 22 468 651

TABLE 5 Mass Differences of VP2 among 13 AAV Isotypes AAV1 AAV2 395 AAV2AAV3 226 169 AAV3 AAV4 467 862 693 AAV4 AAV5 810 1205 1036 343 AAV5 AAV62 392 224 470 812 AAV6 AAV7 278 116 52 746 1088 276 AAV7 AAV8 425 31 199893 1235 423 147 AAV8 AAV9 117 278 109 584 927 115 161 308 AAV9 AAV10177 217 49 645 987 175 101 248 60 AAV10 AAV11 299 694 525 168 511 301578 725 416 476 AAV11 AAV12 812 417 586 1279 1622 810 533 386 695 6351111 AAV12 AAVRh10 160 235 66 627 970 157 119 266 43 18 459 652

TABLE 6 Mass Differences of VP3 among 13 AAV Isotypes AAV1 AAV2 457 AAV2AAV3 332 125 AAV3 AAV4 12 445 320 AAV4 AAV5 54 511 386 66 AAV5 AAV6 2455 330 10 56 AAV6 AAV7 416 673 748 428 362 418 AAV7 AAV8 288 169 44 276342 286 704 AAV8 AAV9 216 241 116 204 270 214 632 72 AAV9 AAV10 121 336211 109 175 119 537 167 95 AAV10 AAV11 179 278 153 167 233 177 595 10937 58 AAV11 AAV12 329 128 3 317 383 327 745 41 113 208 150 AAV12 AAVRh10117 340 215 105 171 115 533 171 99 4 62 212

No masses within 10 Da of all three VPs between two isotypes areobserved. Even though both VP2 and VP3 have only a 2 Da differencebetween AAV1 and AAV6, the mass difference of VP1 between AAV1 and AAV6is 36, significant enough to be distinguished by an accurate massmeasurement. Therefore, intact protein measurement of VP1, VP2 and VP3is highly specific as an identity test.

These results demonstrate that intact protein analysis and LC/MS/MS canbe used to profile VPs to monitor VP expressions, posttranslationalmodifications, and truncations and to ensure product consistency duringVLP production. These two analyses can also be used to confirmsite-direct mutagenesis or structural characterization for capsidprotein engineering applications.

Example 2: The Role of N Terminal Acetylation of AAV Capsid Proteins

Chemical modifications of cellular proteins are a common means ofcontrolling their functions (Arnesen, T. (2006) Virology 353(2):283-293). N-terminal acetylation (Nt-acetylation), which involves thetransfer of an acetyl group from acetyl coenzyme A to the α-amino groupof the first amino acid residue of a protein (Brown, J. L. and Roberts,W. K. (1976) J Biol Chem 251: 1009-1014; Arnesen, T. et al. (2009) ProcNatl Acad Sci USA 106: 8157-8162), is among the most abundant of proteinmodifications. Unlike most other protein modifications, Nt-acetylationis irreversible; it occurs mainly during the synthesis of the protein,catalyzed by N-terminal acetyltransferases (NATs) associated withribosomes (Gautschi, M. et al. (2003) Mol Cell Biol 23: 7403-7414;Pestana, A. and Pitot, H. C. (1975) Biochemistry 14: 1404-1412;Polevoda, B. et al. (2003) J Biol Chem 278: 30686-97). There are severaldistinct NATs in eukaryotes—NatA-NatF—each composed of one or moresubunits and each acetylating a specific subgroup of N-termini dependingon the amino acid sequence of the first few amino acids (Jornvall, H.(1975) J Theor Biol 55: 1-12; Persson, B. et al. (1985) Eur J Biochem152: 523-527).

Experimental data indicate that proteins with acetylated N-termini aremore stable in vivo than non-acetylated proteins; i.e., Nt-acetylationprotects proteins from degradation (Hershko, A. et al. (1984) Proc NatlAcad Sci USA 81: 7021-7025). One explanation for this might be thediscovery in 2004 that another N-terminal modification, ubiquitination,which involves direct attachment of the small protein ubiquitin to theN-terminal amino acid residue, promotes the subsequent degradation ofthe protein (Ben Saadon, R. et al. (2004) J Biol Chem 279: 41414-41421).Conversely, the Nt-acetylation signals can also be part of a qualitycontrol mechanism to degrade unfolded or misfolded proteins and toregulate in vivo protein stoichiometries (Hwang, C. S. et al. (2010)Science 327: 973-977).

A systematic analysis of the predicted N-terminal processing ofcytosolic proteins, versus those destined to be sorted to the secretorypathway, revealed that the cytosolic proteins were profoundly biased infavor of processing, but there is an equal and opposite bias againstsuch modification for secretory proteins (Forte, G. M. A. et al. (2011)PLoS Biology, 4 May 2011 Volume 9). Mutations in secretory signalsequences that lead to their acetylation result in mis-sorting to thecytosol in a manner that is dependent upon the N-terminal processingmachinery. Hence N-terminal acetylation represents an early determiningstep in the cellular sorting of nascent polypeptides that represent anextra layer of stringency in order to ensure that proteins destined toremain in the cytosol actually reside in the cytosol. The eukaryoticcell comprises several distinct compartments, called organelles,required to perform specific functions. The proteins in thesecompartments are synthesized in the cytoplasm and so require complexsorting mechanisms to ensure their delivery to the appropriateorganelle. Proteins are modified by acetylation of their amino terminusat a very early stage in their synthesis. There is a profound differencebetween the likelihood of such a modification on cytoplasmic proteinsand on those destined for one of the major organelles, the endoplasmicreticulum (ER): whereas cytoplasmic proteins are typically acetylated,those bound for the ER are largely unmodified. Moreover, when specificER proteins are engineered to induce their acetylation their targetingto the ER was inhibited (Forte, G. M. A. et al. (2011) PLoS Biology, 4May 2011 Volume 9).

The contractile proteins actin and tropomyosin have been shown torequire NatB-mediated Nt-acetylation for proper function, specificallyinvolving actin-tropomyosin binding and actomyosin regulation (Coulton,A. T. et al. (2010) J Cell Sci 123: 3235-3243; Polevoda, B. et al.(2003) J Biol Chem 278: 30686-97). Thus Nt acetylation of AAV capsidproteins may have importance in the transduction potential of rAAVvectors. If AAV vectors fail to gain entry into the nucleus, theyconsequently fail to transduce cells. The role of actin filaments andFKBP52 (FK506-binding protein p52) in the translocation of AAV capsidsfrom the endosome to the nucleus is well defined (Zhao, W. et al. (2006)Virology 353(2): 283-293), Importantly, Nt-acetylation is essential forthe functioning of actin filaments by modulating protein-proteininteractions (Coulton, A. T. et al. (2010) J Cell Sci 123: 3235-3243;Polevoda, B. et al. (2003) J Biol Chem 278: 30686-97).

Though N-terminal acetylation of proteins is a widely known phenomenon,the biological significance of Nt-acetylation on AAV capsid proteins isnot well understood. The predicted N-termini of VP1 and VP3 based on DNAsequencing are both methionine followed by alanine. It has been reportedthat removal of N-terminal methionine by Met-aminopeptidases frequentlyleads to Nt-acetylation of the resulting N-terminal alanine, valine,serine, threonine, and cysteine residues and that the acetylation of theN-terminus acts as a potential degradation signal [21]. Ubiquitinationof viral capsid proteins was suggested as a potential signal forprocessing of the capsid at the time of virion disassembly [22]. Thelink between N-acetylation of VP1 and VP3 and viral capsid degradationand uncoating before the nuclear entry is further investigated.

To understand the functional implications of N-terminal acetylation withregard to AAV capsid proteins, site-directed mutagenesis of VP3N-terminal initiation codons is used to generate AAV mutants.

Methods

AAV capsid proteins are generated with differing amino acids at the2^(nd) position to the initiating methionine (iMet X) to determine ifNt-acetylation is inhibited or reduced, and the functional consequencesare then measured. The ability of the capsid proteins to be traffickedintra-cellularly and/or to acquire post translational modifications suchas glycosylation is assessed, and whether this ability affects theinfectivity of the assembled AAV particle is subsequently determined. Inaddition, the impact of acetylation on ubiquitination/degradation andtargeting to the lysosome, ER, Golgi, or inner nuclear membrane isdetermined.

For example, to assay trafficking or targeting, AAV particles withcapsid proteins having a mutated 2^(nd) position (e.g., iMet X) arefluorescently labeled and used to infect cells (e.g., HeLa cells). TheseAAV particles are assayed for one or more of: time of viral particleuptake, colocalization of AAV particles with specific compartmentalmarkers (e.g., Golgi, ER, or lysosomal proteins or other markers),nuclear accumulation (e.g., as assayed by colocalization with a nuclearmarker or stain), and/or sensitivity of trafficking to specificinhibitors of early endosomal escape (such as bafilomycin A or ammoniumchloride), as compared to fluorescently labeled wild-type AAV particlesused to infect the same cell line (see, e.g., Bartlett, J. S. et al.(2000) J. Virol. 74:2777-2785 for a description of such assays).

To assay infectivity, AAV particles with capsid proteins having amutated 2^(nd) position (e.g., iMet X) are used to infect cells (e.g.,HeLa cells), and their transduction efficiency is compared to wild-typeAAV particles (e.g., having the same AAV serotype and infecting the sametype of cells).

To assay glycosylation, AAV particles with capsid proteins having amutated 2^(nd) position (e.g., iMet X) are used to infect cells (e.g.,HeLa cells). AAV particles from infected cells are subjected to one ormore assays including without limitation chemical detection ofglycosylation (e.g., applying a commercially available digoxigenin (DIG)glycan detection and/or fluorescent glycoprotein detection kit ondenatured and electrophoretically separated capsid proteins) and massspectrometry (e.g., FT-ICR MS), as compared to wild-type AAV particlesused to infect the same cell line (see, e.g., Murray, S. et al. (2006)J. Virol. 80:6171-6176 for a description of such assays).

To assay ubiquitination, AAV particles with capsid proteins having amutated 2^(nd) position are used to infect cells (e.g., HeLa cells). AAVparticles are immunoprecipitated from infected cells with an anti-capsidantibody, then subjected to Western blotting with an anti-ubiquitinantibody and compared to wild-type AAV particles used to infect cells inthe same manner. Mutant AAV particles may also be used in in vitroubiquitination assays, as compared to wild-type AAV particles (see,e.g., Yan, Z. et al. (2002) J. Virol. 76:2043-2053).

Example 3: The Role of Deamidation of AAV2 Capsid Proteins

Sequence analysis of the AAV2 capsid protein revealed potentialdeamidation sites, as underlined in the following amino acid sequence:

(SEQ ID NO: 3) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYK

EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL.

In particular, a potential deamidation site is found at N57/G58 in thephospholipase A2 domain (Ca++ binding site), as bolded and italicized inthe above sequence. The following experiments were aimed at exploringwhether deamidation at N57 can lead to reduced potency and/or truncationof AAV2, as well as whether different AAV production methods may havedifferent effects on deamidation. For example, the producer cell linemethod (see Martin et al., (2013) Human Gene Therapy Methods 24:253-269;U.S. PG Pub. No. US2004/0224411; and Liu, X. L. et al. (1999) Gene Ther.6:293-299) may induce a higher level of deamidation at N57, as comparedto the triple transfection method. According to the crystal structure ofAAV2, N57 is not shown; however, N382 and N511 are partially exposed,and N715 is fully exposed.

Methods

Enzymatic Digestions of AAV1 and AAV2 VPs

10 μg of each AAV1-EGFP or AAV2-EGFP material (generated from tripletransfection as well as producer cell line process) were concentratedusing Amicon filters (10 kDa MWCO), denatured with 6 M Guanidine-HCl, 50mM Tris at pH 8.5. The proteins were reduced with 5 mM DTT at 60° C. for30 minutes in darkness, alkylated with 15 mM iodoacetamide at roomtemperature for 30 minutes, and then buffer exchanged into 25 mM Tris pH7.1 for digestion using Bio-Spin® 6 Tris micro-columns. After bufferexchange, the samples were split into two aliquots. Each aliquot wasdigested with trypsin at 1:25 or Asp-N at 1:50 enzyme: protein ratio(wt/wt) for 2 hours at 37° C., respectively.

UPLC/MS/MS Peptide Mapping

The protein digests were also analyzed by UPLC/MS/MS in AcquityUPLC-Xevo qTOF MS. BEH300 C18 column (2.1×150 mm) was used forseparation in the mobile phases with 0.1% formic acid inwater/acetonitrile gradient at a flow rate 0.25 ml/min. The mass spectrawere acquired in the positive MSe resolution mode in the mass range of50-2000.

Determination of Deamidation Levels in AAV VPs

The extracted ion chromatograms (XIC) of peptides containing NG sites(T9, T49, and T67 in AA1 and AAV2 VP) and their corresponding deamidatedspecies were used for calculation of deamidation levels.

In order to compare AAV vectors produced by the triple transfection(TTx) and producer cell line (PCL) methods, AAV1 or AAV2 tagged withEGFP was produced using the TTx or PCL method. Truncated VP1 (tVP1) wasfound to be present in AAV2-EGFP produced by PCL, but not in theAAV2-EGFP produced by TTx. AAV1-EGFP was not found to have tVP1,regardless of the production method. The in vitro potency of AAV2produced by the PCL method was also found to be reduced, as compared toAAV2 produced by TTx. Mutant N57K and N57Q AAV2 particles were alsofound to have reduced potency and disrupted Ca++ binding.

The following table provides the tryptic peptides that were analyzed toexamine each potential deamidation site, as well as the correspondingresidue.

TABLE 7 Tryptic peptides containing NG sitesPeptide (NG sequence underlined) Residue YLGPF NG LDK (SEQ ID NO: 9) N57EVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLP N382 PFPADVFMVPQYGYLTLN NGSQAVGRSSFYCLEYFPSQMLR (SEQ ID NO: 10) YNL NG R (AAV1) (SEQ ID NO: 11)N511 YHL NG R (AAV2) (SEQ ID NO: 12) N511 SANVDFTVDN NGLYTEPR (AAV1) (SEQ ID NO: 13) N715 SVNVDFTVDT NGVYSEPR (AAV2) (SEQ ID NO: 14) N715

As shown in Table 7, the T9 peptide YLGPFNGLDK (SEQ ID NO: 9) was usedto monitor N57, the T38 peptideEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLR(SEQ ID NO: 10) was used to monitor N382, the T49 peptides YNLNGR (SEQID NO: 11) and YHLNGR (SEQ ID NO: 12) were used to monitor N511 in AAV1or AAV2 (respectively), and the T67 peptides SANVDFTVDNNGLYTEPR (SEQ IDNO: 13) and SVNVDFTVDTNGVYSEPR (SEQ ID NO: 14) were used to monitor N715in AAV1 or AAV2 (respectively).

LC/MS/MS analysis was used to compare the percentage of deamidation inAAV1 and AAV2 particles produced by the TTx and PCL methods. The resultsfrom the T9 peptide are shown in FIGS. 6A & 6B. The results from the T49peptide are shown in FIGS. 7A & 7B. The results from the T67 peptide areshown in FIGS. 8A & 8B. These results are summarized in Table 8. The T38peptide was not detected due to its size.

TABLE 8 Summary of LC/MS/MS results % Deamidation N57 N511 N715 AAV1 TTx7.9 30.9 18.1 PCL 11.3 27.4 18.7 AAV2 TTx 6.7 39.6 27.4 PCL 18.4 42.328.0

In particular, AAV2 produced by PCL showed nearly a 3-fold increase indeamidation as compared to AAV2 produced by TTx. These results suggestthat deamidation decreases AAV potency, as the in vitro potency of AAV2produced by PCL is reduced.

Conclusions

Taken together, Examples 1-3 demonstrate methods for analyzing intactproteins of viral particles (e.g., AAV capsid proteins) using LC/MS.Molecular weights were measured accurately, and these techniques may bealso used to assess N-termini and/or modifications of viral capsidproteins. Moreover, these methods are adaptable as capsid serotypeidentity assays useful in gene therapy, e.g., as an analytical platform.These results further establish a correlation between capsid proteinstructure (e.g., truncations, deamidation, etc.) and potency, suggestingthat point mutations at key sites may be used to engineer more effectivevectors.

Example 4: Elucidating the Role of N Terminal Acetylation of AAV CapsidProteins

As discussed above, the N-termini of AAV capsid proteins are highlyconserved across serotypes (FIG. 5 ). The techniques described inExample 1 allow for interrogation of VP expression and posttranslationalmodifications. The role and biological significance of N-terminalacetylation of AAV capsid proteins was next examined.

Results

To elucidate the potential role of deacetylation of AAV capsid proteins,AAV5 deacetylation variants were tested. An AAV5 particle expressingeGFP under the CBA promoter (AAV5-CBA-Egfp) was compared to AAV5variants with the amino acid adjacent to the initiating methionine(iMET) mutated for VP1 and VP3 (deAC-AAV5-CBA-eGFPs). Three amino acidspredicted to have a low likelihood of acetylation by NatA, NatC, or NatDwere chosen for generating variants: Gly, Leu, and Pro, as illustratedin Table 9 below.

TABLE 9 N-terminal acetylation frequency NT-AC N-term aa TransferaseFREQUENCY MET-ALA Nat A High Normally found in VP1 & VP 3 MET-SER Nat AHigh Normally found in VP1 & VP3 for AAV5 AAV variants MET-GLY Nat A LowMET-LEU NatC Low MET-PRO NatD/other Low

The following AAV5 deacetylated (deAC) mutants were generated:

-   -   S2GVP1—Ser changed to Gly at position 2 in AAV5VP1    -   S2LVP1—Ser changed to Leu at position 2 in AAV5VP1    -   S2PVP1—Ser changed to Pro at position 2 in AAV5VP1    -   S2GVP3—Ser changed to Gly at position 2 in AAV5VP3    -   S2LVP3—Ser changed to Leu at position 2 in AAV5VP3    -   S2PVP3—Ser changed to Pro at position 2 in AAV5VP3    -   S2PVP1/VP3—Ser changed to Pro at position 2 in both AAV5 VP1 and        VP3    -   S2GVP1/VP3—Ser changed to Gly at position 2 in both AAV5 VP1 and        VP3    -   S2LVP1/VP3—Ser changed to Leu at position 2 in both AAV5 VP1 and        VP3

These variants were generated using the TTX method as described above.All AAV5 variants showed good productivity, with yields greater than10¹³ total VG. All AAV5 variants also showed the expected VP1:VP2:VP3protein ratio by SYPRO protein gel analysis (FIG. 9 ). Next, LC/MS wasused to confirm that all AAV5 variants had decreased acetylation, asshown in Table 10.

TABLE 10 LC/MS analysis of AAV5 variant acetylation VP1 VP1 Δmass VP2VP2 Δmass VP3 VP3 Δmass mutants Theo. Exp. (VP1) Theo. Exp. (VP2) Theo.Exp. (VP3) note 1 deAC-AAV5 80234 nd 65283 65293 10 59463 59472 9 VP1not (S2GVP1)/ detectable CBA-eGFP 2 deAC-AAV5 80346 80501 181 6528365292 9 59463 59471 8 VP1 incorrect (S2LVP1)/ CBA-eGFP 3 deAC-AAV5 80234nd 65253 65261 8 59391 59398 7 confirmed (S2GVP3)/ CBA-eGFP 4 deAC-AAV580336 80363 27 65309 65309 0 59447 59620 173 VP3 incorrect (S2LVP3)/CBA-eGFP 5 deAC-AAV5 80314 80324 10 65293 65300 7 59431 59438 7confirmed (S2PVP1VP3)/ CBA-eGFP 6 deAC-AAV5 80234 80243 9 65253 65261 859391 59398 7 confirmed (S2GVP1VP3)/ CBA-eGFP 7 deAC-AAV5 80336 80346 1065293 65292 1 59431 59430 1 confirmed (S2PVP3)/ CBA-eGFP 8 deAC-AAV580314 80313 1 65283 65291 8 59463 59470 7 confirmed (S2PVP1)/ CBA-eGFP 9deAC-AAV5 80346 nd 65309 65318 9 59447 59629 182 VP3 incorrect (S2LVP1VP3)/ CBA-eGFP nd = not determined

These LC/MS analyses confirmed that AAV5 variants were deacetylated. Thevariants S2LVP1, S2LVP3, and S2LVP1/VP3 all showed increased mass(increased from 173 to 182) in VP1 and VP3 proteins, suggesting thatchanging the second N-terminal amino acid to a leucine in VP1 or VP3alters the protein, resulting in an increase in mass.

Next, AAV5 variants were assayed in an in vitro transduction assay usingeGFP as a reporter gene (FIG. 10 ). The assay was designed to evaluatetransduction by AAV5 deacetylated mutant variants at 10⁶ multiplicity ofinfection (MOI), comparing each variant to the parental, unmodified AAV5particle. Three cell lines were used: 293, HuH7, and HeLa cells.Following infection, cells were assayed to determine vector genome copynumber (vg/μg cellular protein) and eGFP expression (by ELISA). Vectorgenome copy number (vg/μg protein) represents the efficiency at whichthe AAV5 variant enters the cell, and eGFP represents the efficiency ofcapsid intracellular trafficking, since transgene expression requiresthe capsid/vector DNA to efficiently traffic to the nucleus (FIG. 10 ).Vector genomes were quantified by TaqMan analysis.

FIG. 11 shows that, based on vector genome analyses, AAV5 deacetylatedmutant vectors infected all three test cell lines at similar, butreduced, levels as compared to the parental unmodified AAV5 particles.FIG. 12 shows that AAV5 deacetylated mutant vectors all resulted inreduced eGFP expression in all three cell lines, as compared totransduction with parental unmodified AAV5.

Conclusions

As predicted, no acetylation was observed in N-terminal Ser toPro/Leu/Gly mutant variants when examined by LC/MS. AAV5 deAC variantsshowed robust vector production, and AAV5 deAC variants infected cellsat levels comparable to parental AAV5. However, functional proteinlevels in cells infected with deAC variants were greatly reduced whencompared to the parental AAV5. These data suggest that tropism isminimally affected by a lack of N-terminal deacetylation in VP1/VP3, butdownstream processing (e.g., trafficking and/or degradation) issignificantly affected. Since the variants tested demonstrated reducedin vitro activity, one of skill in the art may appreciate that variantscharacterized by reduced or eliminated acetylation could be employed,inter alia, when decreased levels of transduction are desirable.

Example 5: Assessment of Deamidation of AAV Capsid Proteins

Examples 1 and 3 demonstrate techniques that allow the interrogation ofpost-translational modifications of AAV capsid proteins and explore therole of deamidation of the AAV2 capsid. The following Example testedwhether deamidation reduces potency and/or induces truncation of capsidproteins, and whether different manufacturing processes can inducedifferent levels of deamidation.

Methods

AAV particles were generated and deamidation status assayed as describedin Example 3.

Results

As described in Example 3, a potential deamidation site is found atN57/G58 in the phospholipase A2 domain (Ca++ binding site) in VP1 of theAAV2 capsid. The N57/G58 motif is conserved across AAV serotypes (FIG.13 ). Example 3 showed that AAV2 produced by PCL exhibited nearly a3-fold increase in deamidation as compared to AAV2 produced by TTx (seeFIGS. 6A & 6B and Table 8).

In examining VP1, VP2, and VP3 production by protein gels, a truncatedVP1 protein (tVP1) was detected only in AAV2 capsid proteins produced bythe PCL method (FIG. 14 ).

A series of AAV2 deamidation mutants was generated next. These mutantstargeted the Gly residue in the canonical NG sequence. Mutationstargeting the A35 residue (see FIG. 13 ), the N-terminal amino acid fortVP1 were also generated, as shown in Table 11. The pAF277 and pAF279mutants bearing multiple mutations did not package.

TABLE 11 Deamidation mutants Name mutation avg drp/cell pAF274 G58K4.54E+03 pAF275 G58D 5.00E+03 pAF276 G58Q 5.41E+03 pAF277 G58, 383, 512,716K 7.2 pAF278 A35N 6.89E+03 pAF279 A35N, G58, 383, 512, 765K 2.2 293 —0.9 PIM45 Control 6.28E+03 K = positive charge (basic) D = negativecharge (acidic) Q = polar

Deamidation of variants were next analyzed by LC/MS as described inExample 3 above. The AAV2A35N and AAV2G58D variants had altereddeamidation as compared to the parental AAV2 (FIG. 15 ). In particular,the AAV2A35N mutant had increased deamidation (17.8%) as compared toparental AAV2 (5.7%). The AAV2G58D variant had reduced deamidation(1.1%) as compared to parental AAV2. SYPRO protein gel analysisdemonstrated that the AAV2 deamidation mutants exhibited the correctVP1:VP2:VP2 ratio (FIG. 16 ).

Next, AAV2 deamidation variants were assayed in an in vitro transductionassay using eGFP as a reporter gene (FIG. 17 ). The assay was designedto evaluate transduction by AAV2 deamidation mutant variants at 10⁶multiplicity of infection (MOI), comparing each variant to the parental,unmodified AAV2 particle. Three cell lines were used: 293, HuH7, andHeLa cells. Following infection, cells were assayed to determine vectorgenome copy number (vg/μg cellular protein) and eGFP expression (byELISA). Vector genome copy number (vg/μg protein) represents theefficiency at which the AAV2 variant enters the cell, and eGFPrepresents the efficiency of capsid intracellular trafficking, sincetransgene expression requires the capsid/vector DNA to efficientlytraffic to the nucleus (FIG. 17 ). Vector genomes were quantified byTaqMan analysis.

Vector genome analysis indicated that AAV2 deamidation mutant variantsinfected all cell lines tested at levels comparable to that of parentalAAV2 vectors (FIG. 18 ). Importantly, the AAV2A35N variant was found tobe more potent than the parental AAV2 vector for transduction in allthree cell lines (FIG. 19 ). The AAV2G58D variant was found to be morepotent than the parental AAV2 vector in HuH7 cells (FIG. 19 ).

Conclusions

In summary, AAV2 deamidation mutant vectors infect cells at levelscomparable to the parent AAV2 particles (e.g., comparable vg/μg cellularprotein). However, based on analysis of eGFP levels in transduced cells,the AAV2A35N variant had higher potency than the parental AAV2 in allcell lines tested, and the AAV2G58D variant had higher potency than theparental AAV2 in HuH7 cells (a liver-derived cell line). These resultssuggest that the A35N mutation may be effective in increasing vectorpotency for transducing many cell types, and that the G58D mutation mayalso be effective in increasing potency in certain cell types, e.g.,liver cells.

REFERENCES

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SEQUENCES

All polypeptide sequences are presented N-terminal to C-terminal unlessotherwise noted. All nucleic sequences are presented 5′ to 3′ unlessotherwise noted.

Nucleotide sequence of potential AAV2 VP3initiation codons (ATG codons underlined) (SEQ ID NO: 1)ATGGCTACAGGCAGTGGCGCACCAATGGCAGACPolypeptide sequence corresponding to potentialAAV2 VP3 initiation codons (methionines underlined) (SEQ ID NO: 2)MATGSGAPMAD AAV2 VP1 polypeptide sequence (SEQ ID NO: 3)MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL VP1 N-terminal tryptic peptide (N-terminal alanineis acetylated) (SEQ ID NO: 4) AADGYLPDWLEDTLSEGIRVP3 N-terminal Asp-N peptide (N-terminal alanine is acetylated)(SEQ ID NO: 5) ATGSGAPM Common VP1 N-terminal sequence (SEQ ID NO: 6)MAADGYLPDWLED Nucleotide sequence of potential AAV7 VP3initiation codons (start codons underlined) (SEQ ID NO: 7)GTGGCTGCAGGCGGTGGCGCACCAATGGCAGACAATAACNucleotide sequence of mutated ITR (SEQ ID NO: 8)CACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCACGCCCGGGCTTTGCCCGGGCG

What is claimed is:
 1. A method to determine the serotype of an adeno-associated virus (AAV) particle comprising a) denaturing the AAV particle, b) directly subjecting the denatured AAV particle to liquid chromatography/mass spectrometry (LC/MS) intact protein analysis, and c) determining the masses of VP1, VP2 and VP3 of the AAV particle; wherein the specific combination of masses of VP1, VP2 and VP3 are indicative of the AAV serotype, and wherein the method is performed in the absence of a gel separation step.
 2. A method to determine the serotype of a viral particle comprising a) denaturing the viral particle, b) directly subjecting the denatured viral particle to liquid chromatography/mass spectrometry (LC/MS) intact protein analysis, and c) determining the masses of one or more capsid proteins of the viral particle; wherein the specific combination of masses of the one or more capsid proteins are indicative of the virus serotype, and wherein the method is performed in the absence of a gel separation step.
 3. The method of claim 1, wherein the calculated masses of VP1, VP2 and VP3 are compared to the theoretical masses of VP1, VP2 and VP3 of one or more AAV serotypes.
 4. The method of claim 1, wherein the AAV particle is denatured with acetic acid, guanidine hydrochloride, and/or an organic solvent.
 5. The method of claim 1, wherein the liquid chromatography is reverse phase liquid chromatography, size exclusion chromatography, hydrophilic interaction liquid chromatography, or cation exchange chromatography.
 6. The method of claim 1, wherein the liquid chromatography is reverse phase chromatography.
 7. The method of claim 6, wherein the reverse phase chromatography is a C4 or C8 reverse chromatography.
 8. The method of claim 1, wherein the liquid chromatography is ultra-performance liquid chromatography (UPLC).
 9. The method of claim 1, wherein the mass spectrometry comprises assisted calibration.
 10. The method of claim 9, wherein sodium iodide is used as a calibrant.
 11. The method of claim 1, wherein the AAV particle is a recombinant AAV (rAAV) particle.
 12. The method of claim 1, wherein the AAV particle comprises an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid, an AAV7 capsid, an AAV8 capsid, an AAVrh8 capsid, an AAV9 capsid, an AAV10 capsid, an AAVrh10 capsid, an AAV11 capsid, an AAV12 capsid, an AAV LK03 capsid, an AAV2R471A capsid, an AAV2/2-7m8 capsid, an AAV DJ capsid, an AAV DJ8 capsid, an AAV2 N587A capsid, an AAV2 E548A capsid, an AAV2 N708A capsid, an AAV V708K capsid, a goat AAV capsid, an AAV1/AAV2 chimeric capsid, a bovine AAV capsid, a mouse AAV capsid rAAV2/HBoV1 (chimeric AAV/human bocavirus virus 1), an AAV2HBKO capsid, an AAVPHP.B capsid or an AAVPHP.eB capsid.
 13. The method of claim 2, wherein the calculated masses of the one or more capsid proteins are compared to the theoretical masses of the one or more capsid proteins of one or more virus serotypes.
 14. The method of claim 2, wherein the liquid chromatography is reverse phase liquid chromatography, size exclusion chromatography, hydrophilic interaction liquid chromatography, or cation exchange chromatography.
 15. The method of claim 2, wherein the liquid chromatography is reverse phase chromatography.
 16. The method of claim 15, wherein the reverse phase chromatography is a C4 or C8 reverse chromatography.
 17. The method of claim 2, wherein the liquid chromatography is ultra-performance liquid chromatography (UPLC).
 18. The method of claim 2, wherein the mass spectrometry comprises assisted calibration.
 19. The method of claim 18, wherein sodium iodide is used as a calibrant.
 20. The method of claim 2, wherein the viral particle comprises a viral vector encoding a heterologous transgene. 